Modified immune cells having enhanced function and methods for screening  for same

ABSTRACT

The present disclosure provides gene edited modified immune cells or precursors thereof (e.g., gene edited modified T cells) comprising an exogenous T cell receptor (TCR) and/or a chimeric antigen receptor (CAR) having specificity for a target antigen, and an insertion and/or deletion in one or more endogenous gene loci, wherein the endogenous gene loci encode regulators of T cell function, thereby resulting in immune cells having enhanced function. Compositions and methods of treatment are also provided. The present invention provides methods of screening for TCR- or CAR-T cells with enhanced immune function (e.g., T cell efficacy, T cell memory, and/or T cell persistence).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/648,722, filed Mar. 27, 2018, which is hereby incorporated byreference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant CA120409awarded by the National Institutes of Health (NIH). The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

The field of adoptive cell therapy is currently comprised of CAR- andTCR-engineered T cells and has emerged from principles of basicimmunology to paradigm-shifting clinical immunotherapy. Adoptive celltherapy of T cells engineered to express artificial receptors thattarget cells of choice has provided an exciting new approach forattacking cancer, and holds equal promise for chronic infection andautoimmunity. Using principles of synthetic biology, advances inimmunology and genetic engineering have made it possible to generatehuman T-cells that display desired specificities and enhancedfunctionalities. For example, clinical trials in patients with advancedB cell leukemias and lymphomas treated with CD19-specific CAR T cellshave induced durable remissions in adults and children.

T cell exhaustion is a state of T cell dysfunction that arises duringmany chronic infections and cancer. It is defined by poor effectorfunction, sustained expression of inhibitory receptors and atranscriptional state distinct from that of functional effector ormemory T cells. Exhaustion prevents optimal control of infection andtumors. Another barrier to efficient T cell based therapy is that Tcells are susceptible to immunosuppression by the microenvironment ofthe targeted cell. For example, PD-L1 in the microenvironment ofprostate cancer cells inhibits the function of TCR- or CAR-engineered Tcells.

Thus, there is a need in the art to identify genes that regulate thefunction of T cells. In particular, there is a need in the art toidentify genes that regulate the function of TCR- or CAR-engineered Tcell function.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that an unbiased, invivo genome-wide screen can identify genes that regulate T cell function(e.g., T cell efficacy, T cell memory, and/or T cell persistence). Thepresent invention provides a method of screening for TCR- or CAR-T cellswith enhanced immune function (e.g., target cell killing). A screeningmethod of the invention resulted in the identification of several genesthat when downregulated, result in TCR- or CAR-T cells with enhancedimmune function. Accordingly, the present invention provides modifiedimmune cells comprising an exogenous TCR and/or CAR, and an insertionand/or deletion in an endogenous gene locus, wherein the endogenous genelocus encodes regulator of T cell function.

In another aspect, a modified immune cell or precursor cell thereof,comprising an insertion and/or deletion in a gene locus encoding for atranscriptional modulator, wherein the insertion and/or deletion iscapable of downregulating gene expression of the endogenoustranscriptional modulator; and an exogenous T cell receptor (TCR) and/orchimeric antigen receptor (CAR) comprising affinity for an antigen on atarget cell, is provided.

In certain exemplary embodiments, the insertion and/or deletion in agene locus is mediated by a CRISPR-related system. In certain exemplaryembodiments, the insertion and/or deletion in a gene locus is mediatedby CRISPR/Cas9. In certain exemplary embodiments, the transcriptionalmodulator is a transcription factor or an epigenetic regulator.

In certain exemplary embodiments, the transcription factor is SIX2 orKLF4. In certain exemplary embodiments, the transcription factor isSIX2. In certain exemplary embodiments, the insertion and/or deletion inthe gene locus encoding for SIX2 is capable of downregulating expressionof SIX2, and/or downregulating gene expression of one or more downstreamtargets of SIX2. In certain exemplary embodiments, the transcriptionfactor is KLF4. In certain exemplary embodiments, the insertion and/ordeletion in the gene locus encoding for KLF4 is capable ofdownregulating expression of KLF4, and/or downregulating gene expressionof one or more downstream targets of KLF4.

In certain exemplary embodiments, the epigenetic regulator is amodulator of histone methylation. In certain exemplary embodiments, themodulator of histone methylation is a component of a histonemethyltransferase complex. In certain exemplary embodiments, thecomponent of a histone methyltransferase complex ishistone-lysine-N-methyltransferase 2D (KMT2D).

In certain exemplary embodiments, the component of a histonemethyltransferase complex is PAGR1. In certain exemplary embodiments,the insertion and/or deletion in the gene locus encoding for PAGR1 iscapable of downregulating gene expression of one or more downstreamtargets of the PAGR1-associated histone methyltransferase complex. Incertain exemplary embodiments, the one or more downstream targets of thePAGR1-associated histone methyltransferase complex is selected from thegroup consisting of ARID1A, ARID3B, ASXL1, DNMT3A, DUSP1, MAP3K8,PAXIP1, PRMT1, SOCS3, and TNFAIP3.

In certain exemplary embodiments, the exogenous TCR is selected from thegroup consisting of a wild-type TCR, a high affinity TCR, and a chimericTCR. In certain exemplary embodiments, the exogenous TCR comprises atleast one disulfide bond. In certain exemplary embodiments, theexogenous TCR comprises a TCR alpha chain and a TCR beta chain.

In certain exemplary embodiments, the exogenous CAR comprises anantigen-binding domain, a transmembrane domain, and an intracellulardomain. In certain exemplary embodiments, the antigen-binding domain isselected from the group consisting of an antibody, an scFv, and a Fab.In certain exemplary embodiments, the exogenous CAR further comprises ahinge domain. In certain exemplary embodiments, the hinge domain isselected from the group consisting of an Fc fragment of an antibody, ahinge region of an antibody, a CH2 region of an antibody, a CH3 regionof an antibody, an artificial hinge domain, a hinge comprising an aminoacid sequence of CD8, or any combination thereof. In certain exemplaryembodiments, the transmembrane domain is selected from the groupconsisting of an artificial hydrophobic sequence and transmembranedomain of a type I transmembrane protein, an alpha, beta, or zeta chainof a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16,CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. In certainexemplary embodiments, the intracellular domain comprises at least oneco-stimulatory domain selected from the group consisting ofco-stimulatory domains of proteins in the TNFR superfamily, CD28, 4-1BB(CD137), OX40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2,CD5, ICAM-1, LFA-1, Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C,and B7-H3. In certain exemplary embodiments, the intracellular domaincomprises an intracellular domain selected from the group consisting ofcytoplasmic signaling domains of a human CD3 zeta chain, FcyRIII, FcsRI,a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-basedactivation motif (ITAM) bearing cytoplasmic receptors, TCR zeta, FcRgamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a,CD79b, and CD66d.

In another aspect, a modified immune cell or precursor cell thereof,comprising: an insertion and/or deletion in one or more gene lociencoding for a protein selected from the group consisting of AZI2,C1orf141, CCDC33, CCL7, CEACAM19, KLF4, MFSD5, PAGR1, SIX2, and USP27X,wherein the insertion and/or deletion is capable of downregulating geneexpression of the one or more endogenous genes; and an exogenous T cellreceptor (TCR) or chimeric antigen receptor (CAR) comprising affinityfor an antigen on a target cell, is provided.

In another aspect, a modified immune cell or precursor cell thereof,comprising: an insertion and/or deletion in one or more gene lociencoding for a protein selected from the group consisting of C1orf141,CCDC33, CCL7, CEACAM19, KLF4, MFSD5, PAGR1, SIX2, and USP27X, whereinthe insertion and/or deletion is capable of downregulating geneexpression of the one or more endogenous genes; and an exogenous T cellreceptor (TCR) and/or chimeric antigen receptor (CAR) comprisingaffinity for an antigen on a target cell, is provided.

In another aspect, a modified immune cell or precursor cell thereof,comprising: an insertion and/or deletion in one or more gene lociencoding for a protein selected from the group consisting of KLF4,PAGR1, and SIX2, wherein the insertion and/or deletion is capable ofdownregulating gene expression of the one or more endogenous genes; andan exogenous T cell receptor (TCR) and/or chimeric antigen receptor(CAR) comprising affinity for an antigen on a target cell, is provided.

In another aspect, a modified immune cell or precursor cell thereof,comprising: an insertion and/or deletion in a gene locus encoding forKLF4, wherein the insertion and/or deletion is capable of downregulatinggene expression of endogenous KLF4; and an exogenous T cell receptor(TCR) and/or chimeric antigen receptor (CAR) comprising affinity for anantigen on a target cell, is provided.

In another aspect, a modified immune cell or precursor cell thereof,comprising: an insertion and/or deletion in a gene locus encoding forSIX2, wherein the insertion and/or deletion is capable of downregulatinggene expression of endogenous SIX2; and an exogenous T cell receptor(TCR) and/or chimeric antigen receptor (CAR) comprising affinity for anantigen on a target cell, is provided.

In another aspect, a modified immune cell or precursor cell thereof,comprising: an insertion and/or deletion in a gene locus encoding forPAGR1, wherein the insertion and/or deletion is capable ofdownregulating gene expression of endogenous PAGR1; and an exogenous Tcell receptor (TCR) and/or chimeric antigen receptor (CAR) comprisingaffinity for an antigen on a target cell, is provided.

In certain exemplary embodiments, the antigen on a target cell is atumor associated antigen (TAA). In some embodiments, the modified cellis an autologous cell. In certain exemplary embodiments, the modifiedcell is derived from a human. In certain exemplary embodiments, themodified cell is a modified T cell.

In another aspect, a method for generating a modified immune cell orprecursor cell thereof, comprising: a) introducing into the immune cella first nucleic acid comprising a nucleic acid sequence encoding anexogenous T cell receptor (TCR) and/or a chimeric antigen receptor (CAR)comprising affinity for an antigen on a target cell; and b) introducinginto the immune cell one or more polypeptides and/or nucleic acidscapable of downregulating gene expression of an endogenoustranscriptional modulator, is provided.

In certain exemplary embodiments, the endogenous transcriptionalmodulator is a transcription factor or an epigenetic regulator. Incertain exemplary embodiments, the transcription factor is SIX2 or KLF4.In certain exemplary embodiments, downregulating gene expression of thetranscription factor results in downregulated gene expression of SIX2,and/or downregulated gene expression of one or more downstream targetsof SIX2. In certain exemplary embodiments, downregulating geneexpression of the transcription factor results in downregulated geneexpression of KLF4, and/or downregulated gene expression of one or moredownstream targets of KLF4.

In certain exemplary embodiments, the epigenetic regulator is amodulator of histone methylation. In certain exemplary embodiments, themodulator of histone methylation is a component of a histonemethyltransferase complex. In certain exemplary embodiments, thecomponent of a histone methyltransferase complex is ahistone-lysine-N-methyltransferase 2D (KMT2D). In certain exemplaryembodiments, the component of a histone methyltransferase complex isPAGR1. In certain exemplary embodiments, downregulating gene expressionof the component of a histone methyltransferase complex results indownregulated gene expression of PAGR1, and/or downregulated geneexpression of one or more downstream targets of the PAGR1-associatedhistone methyltransferase complex. In certain exemplary embodiments, theone or more downstream targets of the PAGR1-associated histonemethyltransferase complex is selected from the group consisting ofARID1A, ARID3B, ASXL1, DNMT3A, DUSP1, MAP3K8, PAXIP1, PRMT1, SOCS3, andTNFAIP3.

In another aspect, a method for generating a modified immune cell orprecursor cell thereof, comprising: a) introducing into the immune cella first nucleic acid comprising a nucleic acid sequence encoding anexogenous T cell receptor (TCR) and/or a chimeric antigen receptor (CAR)comprising affinity for an antigen on a target cell; and b) introducinginto the immune cell one or more polypeptides and/or nucleic acidscapable of downregulating gene expression of one or more endogenousgenes selected from the group consisting of AZI2, C1orf141, CCDC33,CCL7, CEACAM19, KLF4, MFSD5, PAGR1, SIX2, and USP27X, is provided.

In another aspect, a method for generating a modified immune cell orprecursor cell thereof, comprising: a) introducing into the immune cella first nucleic acid comprising a nucleic acid sequence encoding anexogenous T cell receptor (TCR) and/or a chimeric antigen receptor (CAR)comprising affinity for an antigen on a target cell; and b) introducinginto the immune cell one or more polypeptides and/or nucleic acidscapable of downregulating gene expression of one or more endogenousgenes selected from the group consisting of C1orf141, CCDC33, CCL7,CEACAM19, KLF4, MFSD5, PAGR1, SIX2, and USP27X, is provided.

In another aspect, a method for generating a modified immune cell orprecursor cell thereof, comprising: a) introducing into the immune cella first nucleic acid comprising a nucleic acid sequence encoding anexogenous T cell receptor (TCR) and/or a chimeric antigen receptor (CAR)comprising affinity for an antigen on a target cell; and b) introducinginto the immune cell one or more polypeptides and/or nucleic acidscapable of downregulating gene expression of one or more endogenousgenes selected from the group consisting of KLF4, PAGR1, and SIX2, isprovided.

In another aspect, a method for generating a modified immune cell orprecursor cell thereof, comprising: a) introducing into the immune cella first nucleic acid comprising a nucleic acid sequence encoding anexogenous T cell receptor (TCR) and/or a chimeric antigen receptor (CAR)comprising affinity for an antigen on a target cell; and b) introducinginto the immune cell one or more polypeptides and/or nucleic acidscapable of downregulating gene expression of endogenous KLF4, isprovided.

In another aspect, a method for generating a modified immune cell orprecursor cell thereof, comprising: a) introducing into the immune cella first nucleic acid comprising a nucleic acid sequence encoding anexogenous T cell receptor (TCR) and/or a chimeric antigen receptor (CAR)comprising affinity for an antigen on a target cell; and b) introducinginto the immune cell one or more polypeptides and/or nucleic acidscapable of downregulating gene expression of endogenous SIX2, isprovided.

In another aspect, a method for generating a modified immune cell orprecursor cell thereof, comprising: a) introducing into the immune cella first nucleic acid comprising a nucleic acid sequence encoding anexogenous T cell receptor (TCR) and/or a chimeric antigen receptor (CAR)comprising affinity for an antigen on a target cell; and b) introducinginto the immune cell one or more polypeptides and/or nucleic acidscapable of downregulating gene expression of endogenous PAGR1, isprovided.

In certain exemplary embodiments, the first nucleic acid is introducedby viral transduction. In certain exemplary embodiments, the viraltransduction comprises contacting the cell with a viral vectorcomprising the first nucleic acid. In certain exemplary embodiments, theviral vector is selected from the group consisting of a retroviralvector, a lentiviral vector, an adenoviral vector, and anadeno-associated viral vector.

In certain exemplary embodiments, each of the one or more polypeptidesand/or nucleic acids capable of downregulating gene expression comprisesa CRISPR-related system. In certain exemplary embodiments, theCRISPR-related system comprises a CRISPR nuclease and a guide RNA. Incertain exemplary embodiments, the guide RNA comprises a guide sequencethat is sufficiently complementary to a target sequence of an endogenousgene. In certain exemplary embodiments, the guide sequence comprises anucleic acid sequence set forth in any one of SEQ ID NOs: 31-66. Incertain exemplary embodiments, the CRISPR nuclease and the guide RNAcomprise a ribonucleoprotein (RNP) complex.

In certain exemplary embodiments, the target sequence is within thePAGR1 gene and comprises a nucleic acid sequence set forth in any one ofSEQ ID NOs: 31-36. In certain exemplary embodiments, the target sequenceis within the SIX2 gene and wherein the guide RNA comprises a nucleicacid sequence set forth in any one of SEQ ID NOs: 43-48. In certainexemplary embodiments, the target sequence is within the USP27X gene andwherein the guide RNA comprises a nucleic acid sequence set forth in anyone of SEQ ID NOs:49-54. In certain exemplary embodiments, the targetsequence is within the CEACAM19 gene and wherein the guide RNA comprisesa nucleic acid sequence set for the in any one of SEQ ID NOs:55-60. Incertain exemplary embodiments, the target sequence is within theC1orf141 gene and wherein the guide RNA comprises a nucleic acidsequence set forth in any one of SEQ ID NOs: 61-66.

In certain exemplary embodiments, the CRISPR nuclease and/or the guideRNA are encoded by a polynucleotide. In certain exemplary embodiments,the polynucleotide comprises a vector and/or a synthetic mRNA.

In certain exemplary embodiments, each of the one or more polypeptidesand/or nucleic acids capable of downregulating gene expression isintroduced by electroporation. In certain exemplary embodiments, theantigen on a target cell is a tumor associated antigen (TAA). In certainexemplary embodiments, the modified cell is an autologous cell. Incertain exemplary embodiments, the modified cell is derived from ahuman. In certain exemplary embodiments, the modified cell is a modifiedT cell.

In another aspect, a method for enhancing a function of a modifiedimmune cell or precursor cell thereof, wherein the modified cellcomprises an exogenous T cell receptor (TCR) and/or chimeric antigenreceptor (CAR) comprising affinity for an antigen on a target cell,comprising: introducing into the immune cell one or more polypeptidesand/or nucleic acids capable of downregulating gene expression of anendogenous transcriptional modulator, is provided.

In another aspect, a method for enhancing a function of a modifiedimmune cell or precursor cell thereof, wherein the modified cellcomprises an exogenous T cell receptor (TCR) and/or a chimeric antigenreceptor (CAR) comprising affinity for an antigen on a target cell,comprising: introducing into the immune cell one or more polypeptidesand/or nucleic acids capable of downregulating gene expression of one ormore endogenous genes selected from the group consisting of AZI2,C1orf141, CCDC33, CCL7, CEACAM19, KLF4, MFSD5, PAGR1, SIX2, and USP27X,is provided.

In another aspect, a method for enhancing a function of a modifiedimmune cell or precursor cell thereof, wherein the modified cellcomprises an exogenous T cell receptor (TCR) and/or a chimeric antigenreceptor (CAR) comprising affinity for an antigen on a target cell,comprising: introducing into the immune cell one or more polypeptidesand/or nucleic acids capable of downregulating gene expression of one ormore endogenous genes selected from the group consisting of C1orf141,CCDC33, CCL7, CEACAM19, KLF4, MFSD5, PAGR1, SIX2, and USP27X, isprovided.

In another aspect, a method for enhancing a function of a modifiedimmune cell or precursor cell thereof, wherein the modified cellcomprises an exogenous T cell receptor (TCR) and/or a chimeric antigenreceptor (CAR) comprising affinity for an antigen on a target cell,comprising: introducing into the immune cell one or more polypeptidesand/or nucleic acids capable of downregulating gene expression of one ormore endogenous genes selected from the group consisting of KLF4, PAGR1,and SIX2, is provided.

In another aspect, a method for enhancing a function of a modifiedimmune cell or precursor cell thereof, wherein the modified cellcomprises an exogenous T cell receptor (TCR) and/or a chimeric antigenreceptor (CAR) comprising affinity for an antigen on a target cell,comprising: introducing into the immune cell one or more polypeptidesand/or nucleic acids capable of downregulating gene expression ofendogenous KLF4, is provided.

In another aspect, a method for enhancing a function of a modifiedimmune cell or precursor cell thereof, wherein the modified cellcomprises an exogenous T cell receptor (TCR) and/or a chimeric antigenreceptor (CAR) comprising affinity for an antigen on a target cell,comprising: introducing into the immune cell one or more polypeptidesand/or nucleic acids capable of downregulating gene expression ofendogenous SIX2, is provided.

In another aspect, a method for enhancing a function of a modifiedimmune cell or precursor cell thereof, wherein the modified cellcomprises an exogenous T cell receptor (TCR) and/or a chimeric antigenreceptor (CAR) comprising affinity for an antigen on a target cell,comprising: introducing into the immune cell one or more polypeptidesand/or nucleic acids capable of downregulating gene expression ofendogenous PAGR1, is provided.

In certain exemplary embodiments, the function is tumor infiltration. Incertain exemplary embodiments, the function is tumor killing. In certainexemplary embodiments, the function is immunosuppression. In certainexemplary embodiments, the function is resistance to immunosuppressionby PD-1, LAG-3, TIM-3, and/or CTLA-4.

In another aspect, a method for inhibiting activation-induced cell deathof a modified immune cell or precursor cell thereof, wherein themodified cell comprises an exogenous T cell receptor (TCR) and/or achimeric antigen receptor (CAR) comprising affinity for an antigen on atarget cell, comprising: introducing into the immune cell one or morepolypeptides and/or nucleic acids capable of downregulating geneexpression of an endogenous transcriptional modulator, is provided.

In another aspect, a method for inhibiting activation-induced cell deathof a modified immune cell or precursor cell thereof, wherein themodified cell comprises an exogenous T cell receptor (TCR) and/or achimeric antigen receptor (CAR) comprising affinity for an antigen on atarget cell, comprising: introducing into the immune cell one or morepolypeptides and/or nucleic acids capable of downregulating geneexpression of one or more endogenous genes selected from the groupconsisting of AZI2, C1orf141, CCDC33, CCL7, CEACAM19, KLF4, MFSD5,PAGR1, SIX2, and USP27X, is provided.

In another aspect, a method for inhibiting activation-induced cell deathof a modified immune cell or precursor cell thereof, wherein themodified cell comprises an exogenous T cell receptor (TCR) and/or achimeric antigen receptor (CAR) comprising affinity for an antigen on atarget cell, comprising: introducing into the immune cell one or morepolypeptides and/or nucleic acids capable of downregulating geneexpression of one or more endogenous genes selected from the groupconsisting of C1orf141, CCDC33, CCL7, CEACAM19, KLF4, MFSD5, PAGR1,SIX2, and USP27X, is provided.

In another aspect, a method for inhibiting activation-induced cell deathof a modified immune cell or precursor cell thereof, wherein themodified cell comprises an exogenous T cell receptor (TCR) and/or achimeric antigen receptor (CAR) comprising affinity for an antigen on atarget cell, comprising: introducing into the immune cell one or morepolypeptides and/or nucleic acids capable of downregulating geneexpression of one or more endogenous genes selected from the groupconsisting of KLF4, PAGR1, and SIX2, is provided.

In another aspect, a method for inhibiting activation-induced cell deathof a modified immune cell or precursor cell thereof, wherein themodified cell comprises an exogenous T cell receptor (TCR) and/or achimeric antigen receptor (CAR) comprising affinity for an antigen on atarget cell, comprising: introducing into the immune cell one or morepolypeptides and/or nucleic acids capable of downregulating geneexpression of endogenous KLF4, is provided.

In another aspect, a method for inhibiting activation-induced cell deathof a modified immune cell or precursor cell thereof, wherein themodified cell comprises an exogenous T cell receptor (TCR) and/or achimeric antigen receptor (CAR) comprising affinity for an antigen on atarget cell, comprising: introducing into the immune cell one or morepolypeptides and/or nucleic acids capable of downregulating geneexpression of endogenous SIX2, is provided.

In another aspect, a method for inhibiting activation-induced cell deathof a modified immune cell or precursor cell thereof, wherein themodified cell comprises an exogenous T cell receptor (TCR) and/or achimeric antigen receptor (CAR) comprising affinity for an antigen on atarget cell, comprising: introducing into the immune cell one or morepolypeptides and/or nucleic acids capable of downregulating geneexpression of endogenous PAGR1, is provided.

In certain exemplary embodiments, the antigen on a target cell is atumor associated antigen (TAA). In certain exemplary embodiments, themodified cell is an autologous cell. In certain exemplary embodiments,the modified cell is derived from a human. In certain exemplaryembodiments, the modified cell is a modified T cell.

In another aspect, a method for identifying a gene that whendownregulated, results in an enhanced function of an immune cell orprecursor cell thereof, comprising the steps of: a) introducing into aplurality of immune cells a library of nucleic acids encoding for anexogenous T cell receptor (TCR) and/or a chimeric antigen receptor (CAR)having affinity for an antigen, thereby generating a plurality ofmodified immune cells; b) introducing into the plurality of modifiedimmune cells a plurality of agents that target a plurality of endogenousgenes, thereby generating a plurality of edited immune cells; c)contacting the plurality of edited immune cells with a tumor cell; d)selecting one or more edited immune cells that exhibit an enhancedfunction of an immune cell; and e) identifying the endogenous gene thatis downregulated in the one or more edited immune cells of step d),thereby identifying the gene that when downregulated, results in anenhanced function of an immune cell or precursor cell thereof, isprovided.

In certain exemplary embodiments, the steps of a) and b) are carried outsimultaneously. In certain exemplary embodiments, the plurality ofagents in step b) each comprise a nucleic acid encoding a unique guideRNA that targets each of the plurality of endogenous genes. In certainexemplary embodiments, the plurality of agents in step b) each comprisea CRISPR nuclease polypeptide or a nucleic acid that encodes for aCRISPR nuclease. In certain exemplary embodiments, the plurality ofagents in step b) each comprise a CRISPR-related system. In certainexemplary embodiments, the CRISPR-related system comprises a CRISPRnuclease and a guide RNA. In certain exemplary embodiments, the guideRNA comprises a guide sequence that is sufficiently complementary with atarget sequence of a gene that regulates a function of the immune cell.In certain exemplary embodiments, the CRISPR nuclease and the guide RNAcomprise a ribonucleoprotein (RNP) complex. In certain exemplaryembodiments, the identifying step in e) comprises identifying the uniqueguide RNA that targets the downregulated gene.

In another aspect, a method for identifying a gene that whendownregulated, results in an enhanced function of an immune cell orprecursor cell thereof, comprising the steps of: a) introducing into aplurality of immune cells a library of nucleic acids encoding for anexogenous T cell receptor (TCR) and/or a chimeric antigen receptor (CAR)having affinity for an antigen, and encoding for a plurality of guideRNAs that each target a unique region of each of a plurality ofendogenous genes, thereby generating a plurality of modified immunecells; b) introducing into the plurality of modified immune cells aCRISPR nuclease or a nucleic acid that encodes for a CRISPR nuclease,thereby generating a plurality of edited immune cells wherein geneexpression of the plurality of endogenous genes is downregulated; c)contacting the plurality of edited immune cells with a tumor cell; d)selecting one or more edited immune cells that exhibit an enhancedfunction of an immune cell; and e) identifying the endogenous gene thatis downregulated in the one or more edited immune cells of step d),thereby identifying the gene that when downregulated, results in anenhanced function of an immune cell or precursor cell thereof, isprovided.

In certain exemplary embodiments, the step of contacting c) comprisescontacting the plurality of edited immune cells with a tumor cell linecomprising the tumor cell. In certain exemplary embodiments, the step ofcontacting comprises administering the plurality of edited immune cellsinto a tumor-bearing organism comprising the tumor cell.

In another aspect, a nucleic acid library comprising one or more nucleicacids, wherein each of the one or more nucleic acids comprise: a firstnucleic acid encoding for a unique guide RNA; and a second nucleic acidencoding for an exogenous T cell receptor (TCR) and/or chimeric antigenreceptor (CAR) having affinity for an antigen, is provided.

In certain exemplary embodiments, the first nucleic acid comprises guidesequences that are sufficiently complementary with target sequences ofan endogenous gene. In certain exemplary embodiments, each of the one ormore nucleic acids is a vector. In certain exemplary embodiments, eachof the vectors comprise a first expression cassette comprising a firstpromoter operably linked to the first nucleic acid, and a secondexpression cassette comprising a second promoter operably linked to thesecond nucleic acid. In certain exemplary embodiments, the secondexpression cassette further comprises a polynucleotide sequence thatencodes for a selectable marker. In certain exemplary embodiments, theselectable marker is a fluorescent protein.

In another aspect, the modified immune cell or precursor cell thereof ofany one of the preceding aspects and/or embodiments, for use in themethod of any one of the preceding aspects and/or embodiments, isprovided.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings.

FIGS. 1A-1B depict a schematic showing the generation of gene modifiedTCR- or CAR-T cell libraries with the one-shot CRISPR system. FIG. 1Adepicts a schematic of the design of a one-shot CRISPR construct. FIG.1B depicts a schematic illustrating the preparation of a gene modifiedTCR- or CAR-T cell library.

FIG. 2 depicts a schematic of the design of an in vitro genome widescreen using TCR- or CAR-T cell libraries according to an embodiment ofthe present invention.

FIGS. 3A-3B depict a schematic showing the design of an in vivo genomewide screen using TCR- or CAR-T cell libraries according to anembodiment of the present invention. FIG. 3A depicts a schematic showingthe screening of genes that regulate T cell function. FIG. 3B depicts aschematic showing the screening of genes that regulate T cell memory andpersistence.

FIGS. 4A-4B depict graphs showing the fold expansion of CAR-T celllibraries in vitro stimulated with irradiated target tumor cells. FIG.4A depicts a graph showing the fold expansion of a CD19 directed CAR-Tcell library (CAR19). FIG. 4B depicts a graph showing the fold expansionof a PSCA directed CAR-T cell library stimulated with PC3-PSCA tumorcells.

FIGS. 5A-5B depict flow cytometry analysis showing tumor control andfunction of isolated tumor infiltrating lymphocytes. FIG. 5A shows theelimination of Nalm6-GFP tumor cells by CAR19 library T cells. FIG. 5Bshows the upregulation of T cell activation marker CD137 afterco-culture of PC3-PSCA tumor cells with PSCA-CAR library T cells.

FIG. 6 depicts a schematic showing the library and selection process foran in vivo genome wide screen to identify inhibitory pathways of CAR-Tcell therapy.

FIG. 7 depicts the genes identified from deep sequencing and relativeenrichment of single guide RNAs and possible functions of the TOPcandidates.

FIGS. 8A-8B depict top 20 candidates in a PC3-PSCA prostate cancer invivo CRISPR screen. FIG. 8A depicts a heatmap of top 20 potentialtherapeutic targets identified by the CRISPR screen in PC3-PSCA model.FIG. 8B depicts the fold enrichment of the top 20 potential therapeutictargets.

FIG. 9 depicts a heatmap showing the top 20 candidates in a CaPan1pancreatic cancer in vivo CRISPR screen.

FIGS. 10A-10B depict the overlapping top candidates among differenttumors. FIG. 10A depicts the overlapping genes among top 100, 200, and500 hits screen from different tumors.

FIG. 10B depicts fold enrichment of the overlapping candidates.

FIG. 11 depicts a plot of rank and fold change of the TOP candidates.

FIGS. 12A-12C depict the validation of candidates in a PC3-PSCA prostatecancer model. FIG. 12A depicts bioluminescent tumor images of candidatevalidation in the PC3-PSCA prostate cancer model. PC3-PSCA tumors wereestablished in the flank of NSG mice (n=3). After three weeks, the micewere treated with 1×10⁶ WT or KO PSCA CAR-T cells (i.v.). Bioluminescentimaging was conducted before (day 0) and after the mice were treatedwith a single T cell injection. FIG. 12B depicts quantitative imagedata. FIG. 12C depicts tumor volume data.

FIGS. 13A-13C depict the validation of candidates in a CaPan1 pancreaticcancer model. FIG. 13A depicts bioluminescent tumor images of candidatevalidation in the CaPan1 pancreatic cancer model. CaPan1 tumors wereestablished in the flank of NSG mice (n=3). After three weeks, the micewere treated with 1×10⁶ WT or KO PSCA CAR-T cells (i.v.). Bioluminescentimaging was conducted before (day 0) and after the mice were treatedwith a single T cell injection. FIG. 13B depicts quantitative imagedata. FIG. 13C depicts tumor volume data.

FIGS. 14A-14C depict the validation of candidates in an A549-NY-ESO lungcancer model. FIG. 14A depicts bioluminescent tumor images of candidatevalidation in the A549-NY-ESO lung cancer model. A549-NY-ESO tumors wereestablished in the flank of NSG mice (n=3). After one week, the micewere treated with 1×10⁷ WT or KO NY-ESO TCR-T cells (i.v.).Bioluminescent imaging was conducted before (day 0) and after the micewere treated with a single T cell injection. FIG. 14B depictsquantitative image data. FIG. 14C depicts tumor volume data.

FIGS. 15A-15E depict data showing that PAGR1, Klf4, and SIX2 KO enhancein vitro tumor control. FIG. 15A depicts a CD107a assay of PAGR1, Klf4,and SIX2 KO CAR-T cells co-cultured with PC3-PSCA and A375 tumor. FIG.15B depicts a CD107A assay of PAGR1, Klf4, and SIX2 KO CAR-T cellsco-cultured with CaPan1 tumor. FIGS. 15C and 15D depict the killingability of gene knockout CAR-T cells. FIG. 15E depicts results of a CFSEproliferation assay of gene knockout CAR-T cells.

FIGS. 16A-16C depict results showing that PAGR1 and Klf4 KO enhancesPC3-PSCA tumor control. FIG. 16A depicts bioluminescent tumor imaging ofcandidate validation in a PC3-PSCA prostate cancer model. PC3-PSCAtumors were established in the flank of NSG mice (n=3). After threeweeks, the mice were treated with 1×10⁶ WT or KO PSCA CAR-T cells(i.v.). Bioluminescent imaging was conducted before (day 0) and afterthe mice were treated with a single T cell injection. FIG. 16B depictsquantitative image data. FIG. 16C depicts tumor volume data.

FIGS. 17A-17B depict results showing that PAGR1 KO enhances CaPan1 tumorcontrol. FIG. 17A depicts bioluminescent tumor images of candidatevalidation in the CaPan1 pancreatic cancer model. CaPan1 tumors wereestablished in the flank of NSG mice (n=3). After three weeks, the micewere treated with 1×10⁶ WT or KO PSCA CAR-T cells (i.v.). Bioluminescentimaging was conducted before (day 0) and after the mice were treatedwith a single T cell injection. FIG. 17B depicts tumor volume data.

FIGS. 18A-18H depict results showing that PAGR1 enhances CAR-T celltumor accumulation and suppression resistance. FIG. 18A depicts thenumber of tumor infiltrating CAR-T cells in PC3-PSCA model. FIG. 18Bdepicts the number of tumor infiltrating CAR-T cells in CaPan1 model.FIG. 18C depicts the percentage killing of target tumor cells bydifferent CAR-T cells isolated from the tumor. FIGS. 18D-18G depict thelevel of expression of the indicated inhibitory molecule on differenttumor infiltrating CAR-T cells. FIG. 18H depicts results showing thatPAGR1 KO enhances killing ability of the tumor infiltrating cells.

FIGS. 19A-19B depict results showing that PAGR1 KO reduces apoptosis ofCAR-T cells. FIG. 19A depicts plots showing the apoptosis of differentKO CAR-T cells after activation induced cell death. FIG. 19B depictsplots showing the apoptosis of different KO CAR-T cells after co-culturewith target tumor cells.

FIGS. 20A-20D depict results showing that PAGR1 KO enhances CAR-T cellfunction by epigenetic regulation. FIG. 20A depicts Western blotsshowing H3K4 mono-methylation and di-methylation in gene KO T cellsbefore and after bead stimulation. FIG. 20B depicts real-time PCTresults of KMT2D downstream genes in PAGR1 KO and wild type T cells.FIG. 20C depicts results of a ChIP assay of H3K4 mono-methylation onnegative regulators ARID1A and PRMT1. FIG. 20D shows the mRNA level ofpro-survival genes Bcl2 and Myc measured by real-time PCR.

FIG. 21 depicts results showing that PAGR1 KO enhances adoptive T celltherapy in a syngeneic model. Volume of B16-OVA tumor after adoptivetransfer of wild type, PAGR1 and Klf4 KO OT-I T cells. B16-OVA tumorswere established in the flank of C57/BL6 mice (n=4). After eight days,the mice were treated with 1×10⁶ WT or KO OT-I T cells (i.v.). Tumormeasurement conducted before (day 0) and after the mice were treatedwith a single T cell injection.

FIG. 22 depicts results from intracellular staining for DNMT3A performedto confirm knockout efficiencies for selected gRNAs.

FIG. 23 depicts a map of the AAV plasmid that carries the donor templatefor homology-directed repair.

FIG. 24 depicts a schematic of CRISPR/Cas9 and AAV mediated homologousrecombination to knockin GFP into the DNMT3A locus. Boxes representhomologous arms, and the gRNA target locus.

FIG. 25 depicts expression of CD19BBz and EGFP after lentivirus/AAVinfection.

FIG. 26 depicts expression of CD19BBz and EGFP in T cells that weresorted and expanded using the REP protocol described herein.

FIG. 27 depicts results from knockin of EGFP into the DNMT3A gRNA targetregion confirmed by PCR amplification of the junction between DNMT3A DNAand transgene. Boxes represent the homologous arms of DNMT3A and thegRNA target locus. Arrows represent PCR primers.

FIG. 28 depicts results showing that CD107a is expressed at significantlevels in both CD4 and CD8 CD19BBz+KI cells.

FIG. 29 depicts results showing that INFγ is expressed at significantlevels in both CD4 and CD8 CD19BBz+KI cells.

FIG. 30 depicts results showing that TNFα is expressed at significantlevels in both CD4 and CD8 CD19BBz+KI cells.

FIG. 31 depicts results showing that IL-2 is expressed at higher levelsin CD4 CD19BBz+KI cells, but is expressed at lower levels in CD8 cellsthan that in CD19BBz T cells.

FIG. 32 depicts results showing the number of CD8 cells after repeatedstimulations.

FIG. 33 depicts results showing the number of survived cancer cells atthe end of a sequential killing assay.

DETAILED DESCRIPTION

The present invention provides compositions and methods for modifiedimmune cells or precursors thereof (e.g., modified T cells) comprisingan exogenous (e.g., recombinant, transgenic or engineered) T cellreceptor (TCR) and/or chimeric antigen receptor (CAR). In someembodiments, the modified immune cells are genetically edited such thatthe expression of one or more endogenous genes is downregulated. Thesegenetically edited modified immune cells have enhanced immune function.In some embodiments, the genetically edited modified immune cells of thepresent invention are resistant to immunosuppression, e.g., toimmunosuppressive factors of a tumor microenvironment. In certainembodiments, the immune cells have a genetic disruption of a geneencoding an endogenous gene that regulates immune cell function. Incertain embodiments, the endogenous gene negatively regulates immunecell function, such that when the endogenous gene is downregulated, theimmune cell has enhanced immune cell function.

In some embodiments, the provided immune cells, compositions and methodsalter or reduce the effects of T cell inhibitory pathways or signals inthe tumor microenvironment. The modified immune cells of the inventioncounteract the upregulation and/or expression of inhibitory receptors orligands that can negatively control T cell activation and T cellfunction. For example, expression of certain immune checkpoint proteins(e.g., PD-1 or PD-L1) on T cells and/or in the tumor microenvironmentcan reduce the potency and efficacy of adoptive T cell therapy. Suchinhibitory pathways may otherwise impair certain desirable effectorfunctions in the context of adoptive cell therapy. Tumor cells and/orcells in the tumor microenvironment often upregulate certain inhibitoryproteins (such as PD-L1 and PD-L2) delivering an inhibitory signal. Suchproteins may also be upregulated on T cells in the tumormicroenvironment, e.g., on tumor-infiltrating T cells, which can occurfollowing signaling through the antigen receptor (e.g., TCR and/or CAR)or certain other activating signals. Such events may contribute togenetically engineered immune cells (e.g., TCR- or CAR-T cells)acquiring an exhausted phenotype, such as when present in proximity withother cells that express such protein, which in turn can lead to reducedfunctionality. Thus, the modified immune cells of the invention addressthe T cell exhaustion and/or the lack of T cell persistence that is abarrier to the efficacy and therapeutic outcomes of conventionaladoptive cell therapies.

The present invention also provides in vitro and in vivo screeningmethods to identify genes involved in T cell inhibitory pathways orsignals in the tumor microenvironment. The present invention alsoprovides in vitro and in vivo screening methods to identify genes thatregulate T cell memory and persistence. T cell memory is conferred by asubset of T cells called memory T cells. Memory T cells are T cells thathave previously encountered and responded to a target antigen. At asecond encounter of the same target antigen, memory T cells canreproduce to mount a faster and stronger immune response than the firsttime the immune system responded to the target antigen. Certaininhibitory pathways or signals are known in the art to prevent T cellmemory. Thus, the modified immune cells of the present invention mayhave stronger persistence and efficacy.

It is to be understood that the methods described in this disclosure arenot limited to particular methods and experimental conditions disclosedherein as such methods and conditions may vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Furthermore, the experiments described herein, unless otherwiseindicated, use conventional molecular and cellular biological andimmunological techniques within the skill of the art. Such techniquesare well known to the skilled worker, and are explained fully in theliterature. See, e.g., Ausubel, et al., ed., Current Protocols inMolecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008),including all supplements, Molecular Cloning: A Laboratory Manual(Fourth Edition) by MR Green and J. Sambrook and Harlow et al.,Antibodies: A Laboratory Manual, Chapter 14, Cold Spring HarborLaboratory, Cold Spring Harbor (2013, 2nd edition).

A. Definitions

Unless otherwise defined, scientific and technical terms used hereinhave the meanings that are commonly understood by those of ordinaryskill in the art. In the event of any latent ambiguity, definitionsprovided herein take precedent over any dictionary or extrinsicdefinition. Unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular. The useof “or” means “and/or” unless stated otherwise. The use of the term“including,” as well as other forms, such as “includes” and “included,”is not limiting.

Generally, nomenclature used in connection with cell and tissue culture,molecular biology, immunology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein is well-knownand commonly used in the art. The methods and techniques provided hereinare generally performed according to conventional methods well known inthe art and as described in various general and more specific referencesthat are cited and discussed throughout the present specification unlessotherwise indicated. Enzymatic reactions and purification techniques areperformed according to manufacturer's specifications, as commonlyaccomplished in the art or as described herein. The nomenclatures usedin connection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well-known andcommonly used in the art. Standard techniques are used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

That the disclosure may be more readily understood, select terms aredefined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

“Activation,” as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

As used herein, to “alleviate” a disease means reducing the severity ofone or more symptoms of the disease.

The term “antigen” as used herein is defined as a molecule that provokesan immune response. This immune response may involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. The skilled artisan will understand that anymacromolecule, including virtually all proteins or peptides, can serveas an antigen.

Furthermore, antigens can be derived from recombinant or genomic DNA. Askilled artisan will understand that any DNA, which comprises anucleotide sequences or a partial nucleotide sequence encoding a proteinthat elicits an immune response therefore encodes an “antigen” as thatterm is used herein. Furthermore, one skilled in the art will understandthat an antigen need not be encoded solely by a full length nucleotidesequence of a gene. It is readily apparent that the present inventionincludes, but is not limited to, the use of partial nucleotide sequencesof more than one gene and that these nucleotide sequences are arrangedin various combinations to elicit the desired immune response. Moreover,a skilled artisan will understand that an antigen need not be encoded bya “gene” at all. It is readily apparent that an antigen can be generatedsynthesized or can be derived from a biological sample. Such abiological sample can include, but is not limited to a tissue sample, atumor sample, a cell or a biological fluid.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

The term “downregulation” as used herein refers to the decrease orelimination of gene expression of one or more genes.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result or provides a therapeutic orprophylactic benefit. Such results may include, but are not limited toan amount that when administered to a mammal, causes a detectable levelof immune suppression or tolerance compared to the immune responsedetected in the absence of the composition of the invention. The immuneresponse can be readily assessed by a plethora of art-recognizedmethods. The skilled artisan would understand that the amount of thecomposition administered herein varies and can be readily determinedbased on a number of factors such as the disease or condition beingtreated, the age and health and physical condition of the mammal beingtreated, the severity of the disease, the particular compound beingadministered, and the like.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

The term “epitope” as used herein is defined as a small chemicalmolecule on an antigen that can elicit an immune response, inducing Band/or T cell responses. An antigen can have one or more epitopes. Mostantigens have many epitopes; i.e., they are multivalent. In general, anepitope is roughly about 10 amino acids and/or sugars in size.Preferably, the epitope is about 4-18 amino acids, more preferably about5-16 amino acids, and even more most preferably 6-14 amino acids, morepreferably about 7-12, and most preferably about 8-10 amino acids. Oneskilled in the art understands that generally the overallthree-dimensional structure, rather than the specific linear sequence ofthe molecule, is the main criterion of antigenic specificity andtherefore distinguishes one epitope from another. Based on the presentdisclosure, a peptide used in the present invention can be an epitope.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expand” as used herein refers to increasing in number, as inan increase in the number of T cells. In one embodiment, the T cellsthat are expanded ex vivo increase in number relative to the numberoriginally present in the culture. In another embodiment, the T cellsthat are expanded ex vivo increase in number relative to other celltypes in the culture. The term “ex vivo,” as used herein, refers tocells that have been removed from a living organism, (e.g., a human) andpropagated outside the organism (e.g., in a culture dish, test tube, orbioreactor).

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., Sendai viruses, lentiviruses, retroviruses,adenoviruses, and adeno-associated viruses) that incorporate therecombinant polynucleotide.

“Identity” as used herein refers to the subunit sequence identitybetween two polymeric molecules particularly between two amino acidmolecules, such as, between two polypeptide molecules. When two aminoacid sequences have the same residues at the same positions; e.g., if aposition in each of two polypeptide molecules is occupied by anarginine, then they are identical at that position. The identity orextent to which two amino acid sequences have the same residues at thesame positions in an alignment is often expressed as a percentage. Theidentity between two amino acid sequences is a direct function of thenumber of matching or identical positions; e.g., if half (e.g., fivepositions in a polymer ten amino acids in length) of the positions intwo sequences are identical, the two sequences are 50% identical; if 90%of the positions (e.g., 9 of 10), are matched or identical, the twoamino acids sequences are 90% identical.

The term “immune response” as used herein is defined as a cellularresponse to an antigen that occurs when lymphocytes identify antigenicmolecules as foreign and induce the formation of antibodies and/oractivate lymphocytes to remove the antigen.

The term “immunosuppressive” is used herein to refer to reducing overallimmune response.

“Insertion/deletion”, commonly abbreviated “indel,” is a type of geneticpolymorphism in which a specific nucleotide sequence is present(insertion) or absent (deletion) in a genome.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

The term “knockdown” as used herein refers to a decrease in geneexpression of one or more genes.

The term “knockin” as used herein refers to an exogenous nucleic acidsequence that has been inserted into a target sequence (e.g., endogenousgene locus). For example, a CAR/TCR knockin into a target site (e.g.,endogenous gene locus) refers to a nucleic acid sequence encoding achimeric antigen receptor (CAR) or T cell receptor (TCR) that has beeninserted into a target location within the targeted gene sequence. Insome embodiments, where the target sequence is a gene, a knockin isgenerated resulting in the exogenous nucleic acid sequence being inoperable linkage with any upstream and/or downstream regulatory elementscontrolling expression of the target gene. In some embodiments, theknockin is generated resulting in the exogenous nucleic acid sequencenot being in operable linkage with any upstream and/or downstreamregulatory elements controlling expression of the target gene.

The term “knockout” as used herein refers to the ablation of geneexpression of one or more genes.

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

By the term “modified” as used herein, is meant a changed state orstructure of a molecule or cell of the invention. Molecules may bemodified in many ways, including chemically, structurally, andfunctionally. Cells may be modified through the introduction of nucleicacids.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

The term “oligonucleotide” typically refers to short polynucleotides. Itwill be understood that when a nucleotide sequence is represented by aDNA sequence (i.e., A, T, C, G), this also includes an RNA sequence(i.e., A, U, C, G) in which “U” replaces “T.”

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-beta, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). A “subject” or“patient,” as used therein, may be a human or non-human mammal.Non-human mammals include, for example, livestock and pets, such asovine, bovine, porcine, canine, feline and murine mammals. Preferably,the subject is human.

A “target site” or “target sequence” refers to a nucleic acid sequencethat defines a portion of a nucleic acid to which a binding molecule mayspecifically bind under conditions sufficient for binding to occur. Insome embodiments, a target sequence refers to a genomic nucleic acidsequence that defines a portion of a nucleic acid to which a bindingmolecule may specifically bind under conditions sufficient for bindingto occur.

As used herein, the term “T cell receptor” or “TCR” refers to a complexof membrane proteins that participate in the activation of T cells inresponse to the presentation of antigen. The TCR is responsible forrecognizing antigens bound to major histocompatibility complexmolecules. TCR is composed of a heterodimer of an alpha (α) and beta (β)chain, although in some cells the TCR consists of gamma and delta (γ/δ)chains. TCRs may exist in alpha/beta and gamma/delta forms, which arestructurally similar but have distinct anatomical locations andfunctions. Each chain is composed of two extracellular domains, avariable and constant domain. In some embodiments, the TCR may bemodified on any cell comprising a TCR, including, for example, a helperT cell, a cytotoxic T cell, a memory T cell, regulatory T cell, naturalkiller T cell, and gamma delta T cell.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

“Transplant” refers to a biocompatible lattice or a donor tissue, organor cell, to be transplanted. An example of a transplant may include butis not limited to skin cells or tissue, bone marrow, and solid organssuch as heart, pancreas, kidney, lung and liver. A transplant can alsorefer to any material that is to be administered to a host. For example,a transplant can refer to a nucleic acid or a protein.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to, Sendaiviral vectors, adenoviral vectors, adeno-associated virus vectors,retroviral vectors, lentiviral vectors, and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

B. T Cell Receptors

The present invention provides compositions and methods for modifiedimmune cells or precursors thereof (e.g., modified T cells) comprisingan exogenous T cell receptor (TCR). Thus, in some embodiments, thetarget cell has been altered to contain specific T cell receptor (TCR)genes (e.g., a nucleic acid encoding an alpha/beta TCR). TCRs orantigen-binding portions thereof include those that recognize a peptideepitope or T cell epitope of a target polypeptide, such as an antigen ofa tumor, viral or autoimmune protein. In some embodiments, the TCR hasbinding specificity for a tumor associated antigen, e.g., humanNY-ESO-1.

In some embodiments, a modified T cell comprising an exogenous T cellreceptor (TCR) can be used in a screening method of the presentinvention, e.g., to identify genetic targets that when modulated,enhance the function of the TCR-T cell.

A TCR is a disulfide-linked heterodimeric protein comprised of sixdifferent membrane bound chains that participate in the activation of Tcells in response to an antigen. There exists alpha/beta TCRs andgamma/delta TCRs. An alpha/beta TCR comprises a TCR alpha chain and aTCR beta chain. T cells expressing a TCR comprising a TCR alpha chainand a TCR beta chain are commonly referred to as alpha/beta T cells.Gamma/delta TCRs comprise a TCR gamma chain and a TCR delta chain. Tcells expressing a TCR comprising a TCR gamma chain and a TCR deltachain are commonly referred to as gamma/delta T cells. A TCR of thepresent disclosure is a TCR comprising a TCR alpha chain and a TCR betachain.

The TCR alpha chain and the TCR beta chain are each comprised of twoextracellular domains, a variable region and a constant region. The TCRalpha chain variable region and the TCR beta chain variable region arerequired for the affinity of a TCR to a target antigen. Each variableregion comprises three hypervariable or complementarity-determiningregions (CDRs) which provide for binding to a target antigen. Theconstant region of the TCR alpha chain and the constant region of theTCR beta chain are proximal to the cell membrane. A TCR furthercomprises a transmembrane region and a short cytoplasmic tail. CD3molecules are assembled together with the TCR heterodimer. CD3 moleculescomprise a characteristic sequence motif for tyrosine phosphorylation,known as immunoreceptor tyrosine-based activation motifs (ITAMs).Proximal signaling events are mediated through the CD3 molecules, andaccordingly, TCR-CD3 complex interaction plays an important role inmediating cell recognition events.

Stimulation of TCR is triggered by major histocompatibility complexmolecules (MHCs) on antigen presenting cells that present antigenpeptides to T cells and interact with TCRs to induce a series ofintracellular signaling cascades. Engagement of the TCR initiates bothpositive and negative signaling cascades that result in cellularproliferation, cytokine production, and/or activation-induced celldeath.

A TCR of the present invention can be a wild-type TCR, a high affinityTCR, and/or a chimeric TCR. A high affinity TCR may be the result ofmodifications to a wild-type TCR that confers a higher affinity for atarget antigen compared to the wild-type TCR. A high affinity TCR may bean affinity-matured TCR. Methods for modifying TCRs and/or theaffinity-maturation of TCRs are known to those of skill in the art.Techniques for engineering and expressing TCRs include, but are notlimited to, the production of TCR heterodimers which include the nativedisulphide bridge which connects the respective subunits (Garboczi, etal., (1996), Nature 384(6605): 134-41; Garboczi, et al., (1996), JImmunol 157(12): 5403-10; Chang et al., (1994), PNAS USA 91:11408-11412; Davodeau et al., (1993), J. Biol. Chem. 268(21):15455-15460; Golden et al., (1997), J. Imm. Meth. 206: 163-169; U.S.Pat. No. 6,080,840).

In some embodiments, the exogenous TCR is a full TCR or anantigen-binding portion or antigen-binding fragment thereof. In someembodiments, the TCR is an intact or full-length TCR, including TCRs inthe αβ form or γδ form. In some embodiments, the TCR is anantigen-binding portion that is less than a full-length TCR but thatbinds to a specific peptide bound in an MHC molecule, such as binds toan MHC-peptide complex. In some cases, an antigen-binding portion orfragment of a TCR can contain only a portion of the structural domainsof a full-length or intact TCR, but yet is able to bind the peptideepitope, such as MHC-peptide complex, to which the full TCR binds. Insome cases, an antigen-binding portion contains the variable domains ofa TCR, such as variable a chain and variable β chain of a TCR,sufficient to form a binding site for binding to a specific MHC-peptidecomplex. Generally, the variable chains of a TCR contain complementaritydetermining regions (CDRs) involved in recognition of the peptide, MHCand/or MHC-peptide complex.

In some embodiments, the variable domains of the TCR containhypervariable loops, or CDRs, which generally are the primarycontributors to antigen recognition and binding capabilities andspecificity. In some embodiments, a CDR of a TCR or combination thereofforms all or substantially all of the antigen-binding site of a givenTCR molecule. The various CDRs within a variable region of a TCR chaingenerally are separated by framework regions (FRs), which generallydisplay less variability among TCR molecules as compared to the CDRs(see, e.g., Jores et al, Proc. Nat'l Acad. Sci. U.S.A. 87:9138, 1990;Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev.Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the main CDRresponsible for antigen binding or specificity, or is the most importantamong the three CDRs on a given TCR variable region for antigenrecognition, and/or for interaction with the processed peptide portionof the peptide-MHC complex. In some contexts, the CDR1 of the alphachain can interact with the N-terminal part of certain antigenicpeptides. In some contexts, CDR1 of the beta chain can interact with theC-terminal part of the peptide. In some contexts, CDR2 contributes moststrongly to or is the primary CDR responsible for the interaction withor recognition of the MHC portion of the MHC-peptide complex. In someembodiments, the variable region of the β-chain can contain a furtherhypervariable region (CDR4 or HVR4), which generally is involved insuperantigen binding and not antigen recognition (Kotb (1995) ClinicalMicrobiology Reviews, 8:411-426).

In some embodiments, a TCR contains a variable alpha domain (V_(a))and/or a variable beta domain (V_(p)) or antigen-binding fragmentsthereof. In some embodiments, the a-chain and/or β-chain of a TCR alsocan contain a constant domain, a transmembrane domain and/or a shortcytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The ImmuneSystem in Health and Disease, 3 Ed., Current Biology Publications, p.4:33, 1997). In some embodiments, the a chain constant domain is encodedby the TRAC gene (IMGT nomenclature) or is a variant thereof. In someembodiments, the β chain constant region is encoded by TRBC1 or TRBC2genes (IMGT nomenclature) or is a variant thereof. In some embodiments,the constant domain is adjacent to the cell membrane. For example, insome cases, the extracellular portion of the TCR formed by the twochains contains two membrane-proximal constant domains, and twomembrane-distal variable domains, which variable domains each containCDRs.

It is within the level of a skilled artisan to determine or identify thevarious domains or regions of a TCR. In some aspects, residues of a TCRare known or can be identified according to the InternationalImmunogenetics Information System (IMGT) numbering system (see e.g.www.imgt.org; see also, Lefranc et al. (2003) Developmental andComparative Immunology, 2&; 55-77; and The T Cell Factsbook 2nd Edition,Lefranc and LeFranc Academic Press 2001). Using this system, the CDR1sequences within a TCR Va chains and/or νβ chain correspond to the aminoacids present between residue numbers 27-38, inclusive, the CDR2sequences within a TCR Va chain and/or νβ chain correspond to the aminoacids present between residue numbers 56-65, inclusive, and the CDR3sequences within a TCR Va chain and/or νβ chain correspond to the aminoacids present between residue numbers 105-117, inclusive. The IMGTnumbering system should not be construed as limiting in any way, asthere are other numbering systems known to those of skill in the art,and it is within the level of the skilled artisan to use any of thenumbering systems available to identify the various domains or regionsof a TCR.

In some embodiments, the TCR may be a heterodimer of two chains α and β(or optionally γ and δ) that are linked, such as by a disulfide bond ordisulfide bonds. In some embodiments, the constant domain of the TCR maycontain short connecting sequences in which a cysteine residue forms adisulfide bond, thereby linking the two chains of the TCR. In someembodiments, a TCR may have an additional cysteine residue in each ofthe α and β chains, such that the TCR contains two disulfide bonds inthe constant domains. In some embodiments, each of the constant andvariable domains contain disulfide bonds formed by cysteine residues.

In some embodiments, the TCR for engineering cells as described is onegenerated from a known TCR sequence(s), such as sequences of vα,βchains, for which a substantially full-length coding sequence is readilyavailable. Methods for obtaining full-length TCR sequences, including Vchain sequences, from cell sources are well known. In some embodiments,nucleic acids encoding the TCR can be obtained from a variety ofsources, such as by polymerase chain reaction (PCR) amplification ofTCR-encoding nucleic acids within or isolated from a given cell orcells, or synthesis of publicly available TCR DNA sequences. In someembodiments, the TCR is obtained from a biological source, such as fromcells such as from a T cell (e.g. cytotoxic T cell), T-cell hybridomasor other publicly available source. In some embodiments, the T-cells canbe obtained from in vivo isolated cells. In some embodiments, theT-cells can be a cultured T-cell hybridoma or clone. In someembodiments, the TCR or antigen-binding portion thereof can besynthetically generated from knowledge of the sequence of the TCR. Insome embodiments, a high-affinity T cell clone for a target antigen(e.g., a cancer antigen) is identified, isolated from a patient, andintroduced into the cells. In some embodiments, the TCR clone for atarget antigen has been generated in transgenic mice engineered withhuman immune system genes (e.g., the human leukocyte antigen system, orHLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al. (2009) ClinCancer Res. 15: 169-180 and Cohen et al. (2005) J Immunol.175:5799-5808. In some embodiments, phage display is used to isolateTCRs against a target antigen (see, e.g., Varela-Rohena et al. (2008)Nat Med. 14: 1390-1395 and Li (2005) Nat Biotechnol. 23:349-354.

In some embodiments, the TCR or antigen-binding portion thereof is onethat has been modified or engineered. In some embodiments, directedevolution methods are used to generate TCRs with altered properties,such as with higher affinity for a specific MHC-peptide complex. In someembodiments, directed evolution is achieved by display methodsincluding, but not limited to, yeast display (Holler et al. (2003) NatImmunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97,5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54),or T cell display (Chervin et al. (2008) J Immunol Methods, 339,175-84). In some embodiments, display approaches involve engineering, ormodifying, a known, parent or reference TCR. For example, in some cases,a wild-type TCR can be used as a template for producing mutagenized TCRsin which in one or more residues of the CDRs are mutated, and mutantswith an desired altered property, such as higher affinity for a desiredtarget antigen, are selected.

In some embodiments as described, the TCR can contain an introduceddisulfide bond or bonds. In some embodiments, the native disulfide bondsare not present. In some embodiments, the one or more of the nativecysteines (e.g. in the constant domain of the α chain and β chain) thatform a native interchain disulfide bond are substituted with anotherresidue, such as with a serine or alanine. In some embodiments, anintroduced disulfide bond can be formed by mutating non-cysteineresidues on the alpha and beta chains, such as in the constant domain ofthe α chain and β chain, to cysteine. Exemplary non-native disulfidebonds of a TCR are described in published International PCT No.WO2006/000830 and WO2006/037960. In some embodiments, cysteines can beintroduced at residue Thr48 of the α chain and Ser57 of the β chain, atresidue Thr45 of the α chain and Ser77 of the β chain, at residue Tyr10of the α chain and Ser17 of the β chain, at residue Thr45 of the α chainand Asp59 of the β chain and/or at residue Ser15 of the α chain andGlu15 of the β chain. In some embodiments, the presence of non-nativecysteine residues (e.g. resulting in one or more non-native disulfidebonds) in a recombinant TCR can favor production of the desiredrecombinant TCR in a cell in which it is introduced over expression of amismatched TCR pair containing a native TCR chain.

In some embodiments, the TCR chains contain a transmembrane domain. Insome embodiments, the transmembrane domain is positively charged. Insome cases, the TCR chain contains a cytoplasmic tail. In some aspects,each chain (e.g. alpha or beta) of the TCR can possess one N-terminalimmunoglobulin variable domain, one immunoglobulin constant domain, atransmembrane region, and a short cytoplasmic tail at the C-terminalend. In some embodiments, a TCR, for example via the cytoplasmic tail,is associated with invariant proteins of the CD3 complex involved inmediating signal transduction. In some cases, the structure allows theTCR to associate with other molecules like CD3 and subunits thereof. Forexample, a TCR containing constant domains with a transmembrane regionmay anchor the protein in the cell membrane and associate with invariantsubunits of the CD3 signaling apparatus or complex. The intracellulartails of CD3 signaling subunits (e.g. CD3y, CD35, CD3s and CD3ζ chains)contain one or more immunoreceptor tyrosine-based activation motif or ITAM that are involved in the signaling capacity of the TCR complex.

In some embodiments, the TCR is a full-length TCR. In some embodiments,the TCR is an antigen-binding portion. In some embodiments, the TCR is adimeric TCR (dTCR). In some embodiments, the TCR is a single-chain TCR(sc-TCR). A TCR may be cell-bound or in soluble form. In someembodiments, for purposes of the provided methods, the TCR is incell-bound form expressed on the surface of a cell. In some embodimentsa dTCR contains a first polypeptide wherein a sequence corresponding toa TCR a chain variable region sequence is fused to the N terminus of asequence corresponding to a TCR a chain constant region extracellularsequence, and a second polypeptide wherein a sequence corresponding to aTCR β chain variable region sequence is fused to the N terminus asequence corresponding to a TCR β chain constant region extracellularsequence, the first and second polypeptides being linked by a disulfidebond. In some embodiments, the bond can correspond to the nativeinterchain disulfide bond present in native dimeric αβ TCRs. In someembodiments, the interchain disulfide bonds are not present in a nativeTCR. For example, in some embodiments, one or more cysteines can beincorporated into the constant region extracellular sequences of dTCRpolypeptide pair. In some cases, both a native and a non-nativedisulfide bond may be desirable. In some embodiments, the TCR contains atransmembrane sequence to anchor to the membrane. In some embodiments, adTCR contains a TCR α chain containing a variable α domain, a constant αdomain and a first dimerization motif attached to the C-terminus of theconstant a domain, and a TCR β chain comprising a variable β domain, aconstant β domain and a first dimerization motif attached to theC-terminus of the constant β domain, wherein the first and seconddimerization motifs easily interact to form a covalent bond between anamino acid in the first dimerization motif and an amino acid in thesecond dimerization motif linking the TCR α chain and TCR chaintogether.

In some embodiments, the TCR is a scTCR, which is a single amino acidstrand containing an α chain and a β chain that is able to bind toMHC-peptide complexes. Typically, a scTCR can be generated using methodsknown to those of skill in the art, See e.g., International publishedPCT Nos. WO 96/13593, WO 96/18105, WO99/18129, WO04/033685,WO2006/037960, WO2011/044186; U.S. Pat. No. 7,569,664; and Schlueter, C.J. et al. J. Mol. Biol. 256, 859 (1996). In some embodiments, a scTCRcontains a first segment constituted by an amino acid sequencecorresponding to a TCR α chain variable region, a second segmentconstituted by an amino acid sequence corresponding to a TCR β chainvariable region sequence fused to the N terminus of an amino acidsequence corresponding to a TCR β chain constant domain extracellularsequence, and a linker sequence linking the C terminus of the firstsegment to the N terminus of the second segment. In some embodiments, ascTCR contains a first segment constituted by an amino acid sequencecorresponding to a TCR β chain variable region, a second segmentconstituted by an amino acid sequence corresponding to a TCR α chainvariable region sequence fused to the N terminus of an amino acidsequence corresponding to a TCR α chain constant domain extracellularsequence, and a linker sequence linking the C terminus of the firstsegment to the N terminus of the second segment. In some embodiments, ascTCR contains a first segment constituted by an α chain variable regionsequence fused to the N terminus of an α chain extracellular constantdomain sequence, and a second segment constituted by a β chain variableregion sequence fused to the N terminus of a sequence β chainextracellular constant and transmembrane sequence, and, optionally, alinker sequence linking the C terminus of the first segment to the Nterminus of the second segment. In some embodiments, a scTCR contains afirst segment constituted by a TCR β chain variable region sequencefused to the N terminus of a β chain extracellular constant domainsequence, and a second segment constituted by an α chain variable regionsequence fused to the N terminus of a sequence comprising an α chainextracellular constant domain sequence and transmembrane sequence, and,optionally, a linker sequence linking the C terminus of the firstsegment to the N terminus of the second segment. In some embodiments,for the scTCR to bind an MHC-peptide complex, the a and β chains must bepaired so that the variable region sequences thereof are orientated forsuch binding. Various methods of promoting pairing of an α and β in ascTCR are well known in the art. In some embodiments, a linker sequenceis included that links the a and β chains to form the single polypeptidestrand. In some embodiments, the linker should have sufficient length tospan the distance between the C terminus of the α chain and the Nterminus of the β chain, or vice versa, while also ensuring that thelinker length is not so long so that it blocks or reduces bonding of thescTCR to the target peptide-MHC complex. In some embodiments, the linkerof a scTCRs that links the first and second TCR segments can be anylinker capable of forming a single polypeptide strand, while retainingTCR binding specificity. In some embodiments, the linker sequence may,for example, have the formula -P-AA-P-, wherein P is proline and AArepresents an amino acid sequence wherein the amino acids are glycineand serine. In some embodiments, the first and second segments arepaired so that the variable region sequences thereof are orientated forsuch binding. Hence, in some cases, the linker has a sufficient lengthto span the distance between the C terminus of the first segment and theN terminus of the second segment, or vice versa, but is not too long toblock or reduces bonding of the scTCR to the target ligand. In someembodiments, the linker can contain from or from about 10 to 45 aminoacids, such as 10 to 30 amino acids or 26 to 41 amino acids residues,for example 29, 30, 31 or 32 amino acids. In some embodiments, a scTCRcontains a disulfide bond between residues of the single amino acidstrand, which, in some cases, can promote stability of the pairingbetween the α and β regions of the single chain molecule (see e.g. U.S.Pat. No. 7,569,664). In some embodiments, the scTCR contains a covalentdisulfide bond linking a residue of the immunoglobulin region of theconstant domain of the α chain to a residue of the immunoglobulin regionof the constant domain of the β chain of the single chain molecule. Insome embodiments, the disulfide bond corresponds to the native disulfidebond present in a native dTCR. In some embodiments, the disulfide bondin a native TCR is not present. In some embodiments, the disulfide bondis an introduced non-native disulfide bond, for example, byincorporating one or more cysteines into the constant regionextracellular sequences of the first and second chain regions of thescTCR polypeptide. Exemplary cysteine mutations include any as describedabove. In some cases, both a native and a non-native disulfide bond maybe present.

In some embodiments, any of the TCRs, including a dTCR or scTCR, can belinked to signaling domains that yield an active TCR on the surface of aT cell. In some embodiments, the TCR is expressed on the surface ofcells. In some embodiments, the TCR does contain a sequencecorresponding to a transmembrane sequence. In some embodiments, thetransmembrane domain can be a Ca or CP transmembrane domain. In someembodiments, the transmembrane domain can be from a non-TCR origin, forexample, a transmembrane region from CD3z, CD28 or B7.1. In someembodiments, the TCR does contain a sequence corresponding tocytoplasmic sequences. In some embodiments, the TCR contains a CD3zsignaling domain. In some embodiments, the TCR is capable of forming aTCR complex with CD3. In some embodiments, the TCR or antigen bindingportion thereof may be a recombinantly produced natural protein ormutated form thereof in which one or more property, such as bindingcharacteristic, has been altered. In some embodiments, a TCR may bederived from one of various animal species, such as human, mouse, rat,or other mammal.

In some embodiments, the TCR comprises affinity to a target antigen on atarget cell. The target antigen may include any type of protein, orepitope thereof, associated with the target cell. For example, the TCRmay comprise affinity to a target antigen on a target cell thatindicates a particular disease state of the target cell. In someembodiments, the target antigen is processed and presented by MHCs.

In one embodiment, the target cell antigen is a New York esophageal-1(NY-ESO-1) peptide. NY-ESO-1 belongs to the cancer-testis (CT) antigengroup of proteins. NY-ESO-1 is a highly immunogenic antigen in vitro andis presented to T cells via the MHC. CTLs recognizing the A2 presentedepitope NY-ESO₁₅₇₋₁₆₅, SLLMWITQC (SEQ ID NO:1), have been grown from theblood and lymph nodes of myeloma patients. T cell clones specific forthis epitope have been shown to kill tumor cells. A high affinity TCRrecognizing the NY-ESO₁₅₇₋₁₆₅ epitope may recognize HLA-A2-positive,NY-ESO-1 positive cell lines (but not to cells that lack either HLA-A2or NY-ESO). Accordingly, a TCR of the present disclosure may be aHLA-A2-restricted NY-ESO-1 (SLLMWITQC; SEQ ID NO:1)-specific TCR. In oneembodiment, an NY-ESO-1 TCR of the present disclosure is a wild-typeNY-ESO-1 TCR. A wild-type NY-ESO-1 TCR may include, without limitation,the 8F NY-ESO-1 TCR (also referred to herein as “8F” or “8F TCR”), andthe 1G4 NY-ESO-1 TCR (also referred to herein as “1G4” or “1G4 TCR”). Inone embodiment, an NY-ESO-1 TCR of the present disclosure is an affinityenhanced 1G4 TCR, also called Ly95. 1G4 TCR and affinity enhanced 1G4TCR is described in U.S. Pat. No. 8,143,376. This should not beconstrued as limiting in any way, as a TCR having affinity for anytarget antigen is suitable for use in a composition or method of thepresent invention. In some embodiments, a modified immune cellcomprising an exogenous T cell receptor (TCR) having affinity forNY-ESO-1 can be used in a screening method of the present invention,e.g., to identify genetic targets that when modulated, enhance thefunction of the NY-ESO-1 TCR-T cell. A genetic target that is identifiedin a screening method of the present invention is not limited to agenetic target that when modified, modulates the function of a TCR-Tcell having affinity for a specific antigen target. A genetic targetthat is identified in a screening method of the present invention may bea genetic target that when modified, modulates the function of any TCR-Tcell (i.e., having affinity for any antigen target). In someembodiments, genetic targets identified in a screening method of thepresent invention comprising the use of a TCR-T cell, may be globalregulators of TCR-T cell function that is independent of the specificityof the TCR comprised therein.

In some embodiments, the genetic target that is identified in ascreening method of the present invention may be a genetic target thatwhen modified, modulates the function of any TCR-T cell (i.e., havingaffinity for any target antigen). In some embodiments, the genetictarget that is identified in a screening method of the present inventionmay be a genetic target that when modified, modulates the function ofany CAR-T cell (i.e., having affinity for any target antigen). In someembodiments, the genetic target may be a regulator of T cell function.As such, the genetic target, when modified, modulates the function ofany TCR- or CAR-T cell having affinity for any target antigen.

Accordingly, a modified immune cell comprising an exogenous TCR and/orCAR may be edited to modify a genetic target that is a regulator of Tcell function. Such modified immune cells may possess enhanced immunecell function (e.g., target cell killing).

C. Chimeric Antigen Receptors

The present invention provides compositions and methods for modifiedimmune cells or precursors thereof, e.g., modified T cells, comprising achimeric antigen receptor (CAR). Thus, in some embodiments, the immunecell has been genetically modified to express the CAR. CARs of thepresent invention comprise an antigen binding domain, a transmembranedomain, a hinge domain, and an intracellular signaling domain.

The antigen binding domain may be operably linked to another domain ofthe CAR, such as the transmembrane domain or the intracellular domain,both described elsewhere herein, for expression in the cell. In oneembodiment, a first nucleic acid sequence encoding the antigen bindingdomain is operably linked to a second nucleic acid encoding atransmembrane domain, and further operably linked to a third a nucleicacid sequence encoding an intracellular domain.

The antigen binding domains described herein can be combined with any ofthe transmembrane domains described herein, any of the intracellulardomains or cytoplasmic domains described herein, or any of the otherdomains described herein that may be included in a CAR of the presentinvention. A subject CAR of the present invention may also include aspacer domain as described herein. In some embodiments, each of theantigen binding domain, transmembrane domain, and intracellular domainis separated by a linker.

Antigen Binding Domain

The antigen binding domain of a CAR is an extracellular region of theCAR for binding to a specific target antigen including proteins,carbohydrates, and glycolipids. In some embodiments, the CAR comprisesaffinity to a target antigen on a target cell. The target antigen mayinclude any type of protein, or epitope thereof, associated with thetarget cell. For example, the CAR may comprise affinity to a targetantigen on a target cell that indicates a particular disease state ofthe target cell.

In one embodiment, the target cell antigen is a prostate stem cellantigen (PSCA). As such, in one embodiment, a CAR of the presentdisclosure has affinity for PSCA on a target cell. In one embodiment,the target cell antigen is CD19. As such, in one embodiment, a CAR ofthe present disclosure has affinity for CD19 on a target cell. Thisshould not be construed as limiting in any way, as a CAR having affinityfor any target antigen is suitable for use in a composition or method ofthe present invention.

As described herein, a CAR of the present disclosure having affinity fora specific target antigen on a target cell may comprise atarget-specific binding domain. In some embodiments, the target-specificbinding domain is a murine target-specific binding domain, e.g., thetarget-specific binding domain is of murine origin. In some embodiments,the target-specific binding domain is a human target-specific bindingdomain, e.g., the target-specific binding domain is of human origin. Inone embodiment, a CAR of the present disclosure having affinity for PSCAon a target cell may comprise a PSCA binding domain. In one embodiment,a CAR of the present disclosure having affinity for CD19 on a targetcell may comprise a CD19 binding domain.

In some embodiments, a CAR of the present disclosure may have affinityfor one or more target antigens on one or more target cells. In someembodiments, a CAR may have affinity for one or more target antigens ona target cell. In such embodiments, the CAR is a bispecific CAR, or amultispecific CAR. In some embodiments, the CAR comprises one or moretarget-specific binding domains that confer affinity for one or moretarget antigens. In some embodiments, the CAR comprises one or moretarget-specific binding domains that confer affinity for the same targetantigen. For example, a CAR comprising one or more target-specificbinding domains having affinity for the same target antigen could binddistinct epitopes of the target antigen. When a plurality oftarget-specific binding domains is present in a CAR, the binding domainsmay be arranged in tandem and may be separated by linker peptides. Forexample, in a CAR comprising two target-specific binding domains, thebinding domains are connected to each other covalently on a singlepolypeptide chain, through an oligo- or polypeptide linker, an Fc hingeregion, or a membrane hinge region.

In some embodiments, the antigen binding domain is selected from thegroup consisting of an antibody, an antigen binding fragment (Fab), anda single-chain variable fragment (scFv). In some embodiments, a PSCAbinding domain of the present invention is selected from the groupconsisting of a PSCA-specific antibody, a PSCA-specific Fab, and aPSCA-specific scFv. In one embodiment, a PSCA binding domain is aPSCA-specific antibody. In one embodiment, a PSCA binding domain is aPSCA-specific Fab. In one embodiment, a PSCA binding domain is aPSCA-specific scFv. In some embodiments, a PSCA binding domain of thepresent invention is selected from the group consisting of aCD19-specific antibody, a CD19-specific Fab, and a CD19-specific scFv.In one embodiment, a CD19binding domain is a CD19-specific antibody. Inone embodiment, a CD19 binding domain is a CD19-specific Fab. In oneembodiment, a CD19 binding domain is a CD19-specific scFv.

The antigen binding domain can include any domain that binds to theantigen and may include, but is not limited to, a monoclonal antibody, apolyclonal antibody, a synthetic antibody, a human antibody, a humanizedantibody, a non-human antibody, and any fragment thereof. In someembodiments, the antigen binding domain portion comprises a mammalianantibody or a fragment thereof. The choice of antigen binding domain maydepend upon the type and number of antigens that are present on thesurface of a target cell.

As used herein, the term “single-chain variable fragment” or “scFv” is afusion protein of the variable regions of the heavy (VH) and lightchains (VL) of an immunoglobulin (e.g., mouse or human) covalentlylinked to form a VH::VL heterodimer. The heavy (VH) and light chains(VL) are either joined directly or joined by a peptide-encoding linker,which connects the N-terminus of the VH with the C-terminus of the VL,or the C-terminus of the VH with the N-terminus of the VL. In someembodiments, the antigen binding domain (e.g., PSCA binding domain)comprises an scFv having the configuration from N-terminus toC-terminus, VH-linker-VL. In some embodiments, the antigen bindingdomain comprises an scFv having the configuration from N-terminus toC-terminus, VL-linker-VH. Those of skill in the art would be able toselect the appropriate configuration for use in the present invention.

The linker is usually rich in glycine for flexibility, as well as serineor threonine for solubility. The linker can link the heavy chainvariable region and the light chain variable region of the extracellularantigen-binding domain. Non-limiting examples of linkers are disclosedin Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO 2014/087010,the contents of which are hereby incorporated by reference in theirentireties. Various linker sequences are known in the art, including,without limitation, glycine serine (GS) linkers such as (GS)_(n)(GSGGS)_(n) (SEQ ID NO:2), (GGGS)_(n) (SEQ ID NO:3), and (GGGGS)_(n)(SEQ ID NO:4), where n represents an integer of at least 1. Exemplarylinker sequences can comprise amino acid sequences including, withoutlimitation, GGSG (SEQ ID NO:5), GGSGG (SEQ ID NO:6), GSGSG (SEQ IDNO:7), GSGGG (SEQ ID NO:8), GGGSG (SEQ ID NO:9), GSSSG (SEQ ID NO:10),GGGGS (SEQ ID NO:11), GGGGSGGGGSGGGGS (SEQ ID NO:12) and the like. Thoseof skill in the art would be able to select the appropriate linkersequence for use in the present invention. In one embodiment, an antigenbinding domain of the present invention comprises a heavy chain variableregion (VH) and a light chain variable region (VL), wherein the VH andVL is separated by the linker sequence having the amino acid sequenceGGGGSGGGGSGGGGS (SEQ ID NO:12), which may be encoded by the nucleic acidsequence

(SEQ ID NO: 13) GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGTGGCGGCGGATCT.

Despite removal of the constant regions and the introduction of alinker, scFv proteins retain the specificity of the originalimmunoglobulin. Single chain Fv polypeptide antibodies can be expressedfrom a nucleic acid comprising VH- and VL-encoding sequences asdescribed by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883,1988). See, also, U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,956,778; andU.S. Patent Publication Nos. 20050196754 and 20050196754. AntagonisticscFvs having inhibitory activity have been described (see, e.g., Zhao etal., Hyrbidoma (Larchmt) 2008 27(6):455-51; Peter et al., J CachexiaSarcopenia Muscle 2012 Aug. 12; Shieh et al., J Imunol 2009183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63;Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al.,Immunotechnology 1997 3(3):173-84; Moosmayer et al., Ther Immunol 19952(10:31-40). Agonistic scFvs having stimulatory activity have beendescribed (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7;Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit RevImmunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 20031638(3):257-66).

As used herein, “Fab” refers to a fragment of an antibody structure thatbinds to an antigen but is monovalent and does not have a Fc portion,for example, an antibody digested by the enzyme papain yields two Fabfragments and an Fc fragment (e.g., a heavy (H) chain constant region;Fc region that does not bind to an antigen).

As used herein, “F(ab′)2” refers to an antibody fragment generated bypepsin digestion of whole IgG antibodies, wherein this fragment has twoantigen binding (ab′) (bivalent) regions, wherein each (ab′) regioncomprises two separate amino acid chains, a part of a H chain and alight (L) chain linked by an S—S bond for binding an antigen and wherethe remaining H chain portions are linked together. A “F(ab′)2” fragmentcan be split into two individual Fab′ fragments.

In some embodiments, the antigen binding domain may be derived from thesame species in which the CAR will ultimately be used. For example, foruse in humans, the antigen binding domain of the CAR may comprise ahuman antibody or a fragment thereof. In some embodiments, the antigenbinding domain may be derived from a different species in which the CARwill ultimately be used. For example, for use in humans, the antigenbinding domain of the CAR may comprise a murine antibody or a fragmentthereof.

Transmembrane Domain

CARs of the present invention may comprise a transmembrane domain thatconnects the antigen binding domain of the CAR to the intracellulardomain of the CAR. The transmembrane domain of a subject CAR is a regionthat is capable of spanning the plasma membrane of a cell (e.g., animmune cell or precursor thereof). The transmembrane domain is forinsertion into a cell membrane, e.g., a eukaryotic cell membrane. Insome embodiments, the transmembrane domain is interposed between theantigen binding domain and the intracellular domain of a CAR. In someembodiments, the transmembrane domain is naturally associated with oneor more of the domains in the CAR. In some embodiments, thetransmembrane domain can be selected or modified by one or more aminoacid substitutions to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins, to minimizeinteractions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein, e.g., a Type Itransmembrane protein. Where the source is synthetic, the transmembranedomain may be any artificial sequence that facilitates insertion of theCAR into a cell membrane, e.g., an artificial hydrophobic sequence.Examples of the transmembrane domain of particular use in this inventioninclude, without limitation, transmembrane domains derived from (i.e.comprise at least the transmembrane region(s) of) the alpha, beta orzeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40),CD137 (4-1BB), CD154 (CD40L), Toll-like receptor 1 (TLR1), TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9. In some embodiments, thetransmembrane domain may be synthetic, in which case it will comprisepredominantly hydrophobic residues such as leucine and valine.Preferably a triplet of phenylalanine, tryptophan and valine will befound at each end of a synthetic transmembrane domain.

The transmembrane domains described herein can be combined with any ofthe antigen binding domains described herein, any of the intracellulardomains described herein, or any of the other domains described hereinthat may be included in a subject CAR.

In some embodiments, the transmembrane domain further comprises a hingeregion. A subject CAR of the present invention may also include a hingeregion. The hinge region of the CAR is a hydrophilic region which islocated between the antigen binding domain and the transmembrane domain.In some embodiments, this domain facilitates proper protein folding forthe CAR. The hinge region is an optional component for the CAR. Thehinge region may include a domain selected from Fc fragments ofantibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3regions of antibodies, artificial hinge sequences or combinationsthereof. Examples of hinge regions include, without limitation, a CD8ahinge, artificial hinges made of polypeptides which may be as small as,three glycines (Gly), as well as CH1 and CH3 domains of IgGs (such ashuman IgG4).

In some embodiments, a subject CAR of the present disclosure includes ahinge region that connects the antigen binding domain with thetransmembrane domain, which, in turn, connects to the intracellulardomain. The hinge region is preferably capable of supporting the antigenbinding domain to recognize and bind to the target antigen on the targetcells (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2):125-135). In some embodiments, the hinge region is a flexible domain,thus allowing the antigen binding domain to have a structure tooptimally recognize the specific structure and density of the targetantigens on a cell such as tumor cell (Hudecek et al., supra). Theflexibility of the hinge region permits the hinge region to adopt manydifferent conformations.

In some embodiments, the hinge region is an immunoglobulin heavy chainhinge region. In some embodiments, the hinge region is a hinge regionpolypeptide derived from a receptor (e.g., a CD8-derived hinge region).

The hinge region can have a length of from about 4 amino acids to about50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aato about 15 aa, from about 15 aa to about 20 aa, from about 20 aa toabout 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about40 aa, or from about 40 aa to about 50 aa. In some embodiments, thehinge region can have a length of greater than 5 aa, greater than 10 aa,greater than 15 aa, greater than 20 aa, greater than 25 aa, greater than30 aa, greater than 35 aa, greater than 40 aa, greater than 45 aa,greater than 50 aa, greater than 55 aa, or more.

Suitable hinge regions can be readily selected and can be of any of anumber of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acidsto 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitablehinge regions can have a length of greater than 20 amino acids (e.g.,30, 40, 50, 60 or more amino acids).

For example, hinge regions include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), (GSGGS)_(n)(SEQ ID NO:2) and (GGGS)_(n) (SEQ ID NO:3), where n is an integer of atleast one), glycine-alanine polymers, alanine-serine polymers, and otherflexible linkers known in the art. Glycine and glycine-serine polymerscan be used; both Gly and Ser are relatively unstructured, and thereforecan serve as a neutral tether between components. Glycine polymers canbe used; glycine accesses significantly more phi-psi space than evenalanine, and is much less restricted than residues with longer sidechains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2:73-142). Exemplary hinge regions can comprise amino acid sequencesincluding, but not limited to, GGSG (SEQ ID NO:5), GGSGG (SEQ ID NO:6),GSGSG (SEQ ID NO:7), GSGGG (SEQ ID NO:8), GGGSG (SEQ ID NO:9), GSSSG(SEQ ID NO:10), and the like.

In some embodiments, the hinge region is an immunoglobulin heavy chainhinge region. Immunoglobulin hinge region amino acid sequences are knownin the art; see, e.g., Tan et al., Proc. Natl. Acad. Sci. USA (1990)87(1):162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4):1779-1789. As non-limiting examples, an immunoglobulin hinge region caninclude one of the following amino acid sequences: DKTHT (SEQ ID NO:14);CPPC (SEQ ID NO:15); CPEPKSCDTPPPCPR (SEQ ID NO:16) (see, e.g., Glaseret al., J. Biol. Chem. (2005) 280:41494-41503); ELKTPLGDTTHT (SEQ IDNO:17); KSCDKTHTCP (SEQ ID NO:18); KCCVDCP (SEQ ID NO:19); KYGPPCP (SEQID NO:20); EPKSCDKTHTCPPCP (SEQ ID NO:21) (human IgG1 hinge);ERKCCVECPPCP (SEQ ID NO:22) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQID NO:23) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO:24) (human IgG4hinge); and the like.

The hinge region can comprise an amino acid sequence of a human IgG1,IgG2, IgG3, or IgG4, hinge region. In one embodiment, the hinge regioncan include one or more amino acid substitutions and/or insertionsand/or deletions compared to a wild-type (naturally-occurring) hingeregion. For example, His229 of human IgG1 hinge can be substituted withTyr, so that the hinge region comprises the sequence EPKSCDKTYTCPPCP(SEQ ID NO:25); see, e.g., Yan et al., J. Biol. Chem. (2012) 287:5891-5897. In one embodiment, the hinge region can comprise an aminoacid sequence derived from human CD8, or a variant thereof.

Intracellular Signaling Domain

A subject CAR of the present invention also includes an intracellularsignaling domain. The terms “intracellular signaling domain” and“intracellular domain” is used interchangeably herein. The intracellularsignaling domain of the CAR is responsible for activation of at leastone of the effector functions of the cell in which the CAR is expressed(e.g., immune cell). The intracellular signaling domain transduces theeffector function signal and directs the cell (e.g., immune cell) toperform its specialized function, e.g., harming and/or destroying atarget cell.

Examples of an intracellular domain for use in the invention include,but are not limited to, the cytoplasmic portion of a surface receptor,co-stimulatory molecule, and any molecule that acts in concert toinitiate signal transduction in the T cell, as well as any derivative orvariant of these elements and any synthetic sequence that has the samefunctional capability.

Examples of the intracellular signaling domain include, withoutlimitation, the ζ chain of the T cell receptor complex or any of itshomologs, e.g., η chain, FcsRIγ and β chains, MB 1 (Iga) chain, B29 (Ig)chain, etc., human CD3 zeta chain, CD3 polypeptides (Δ, δ and ε), sykfamily tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases(Lck, Fyn, Lyn, etc.), and other molecules involved in T celltransduction, such as CD2, CD5 and CD28. In one embodiment, theintracellular signaling domain may be human CD3 zeta chain, FcyRIII,FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptortyrosine-based activation motif (ITAM) bearing cytoplasmic receptors,and combinations thereof.

In one embodiment, the intracellular signaling domain of the CARincludes any portion of one or more co-stimulatory molecules, such as atleast one signaling domain from CD2, CD3, CD8, CD27, CD28, ICOS, 4-1BB,PD-1, any derivative or variant thereof, any synthetic sequence thereofthat has the same functional capability, and any combination thereof.

Other examples of the intracellular domain include a fragment or domainfrom one or more molecules or receptors including, but not limited to,TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcRgamma, FcR beta (Fc Epsilon RIb), CD79a, CD79b, Fcgamma RIIa, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), OX9, OX40,CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocytefunction-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, aligand that specifically binds with CD83, CD5, ICAM-1, GITR, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8alpha,CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4,IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL,CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18,LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4),CD84, CD 96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1,CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150,IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76,PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other co-stimulatory moleculesdescribed herein, any derivative, variant, or fragment thereof, anysynthetic sequence of a co-stimulatory molecule that has the samefunctional capability, and any combination thereof.

Additional examples of intracellular domains include, withoutlimitation, intracellular signaling domains of several types of variousother immune signaling receptors, including, but not limited to, first,second, and third generation T cell signaling proteins including CD3, B7family costimulatory, and Tumor Necrosis Factor Receptor (TNFR)superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol.(2015) 33(6): 651-653). Additionally, intracellular signaling domainsmay include signaling domains used by NK and NKT cells (see, e.g.,Hermanson and Kaufman, Front. Immunol. (2015) 6: 195) such as signalingdomains of NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012)189(5): 2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol.(2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

Intracellular signaling domains suitable for use in a subject CAR of thepresent invention include any desired signaling domain that provides adistinct and detectable signal (e.g., increased production of one ormore cytokines by the cell; change in transcription of a target gene;change in activity of a protein; change in cell behavior, e.g., celldeath; cellular proliferation; cellular differentiation; cell survival;modulation of cellular signaling responses; etc.) in response toactivation of the CAR (i.e., activated by antigen and dimerizing agent).In some embodiments, the intracellular signaling domain includes atleast one (e.g., one, two, three, four, five, six, etc.) ITAM motifs asdescribed below. In some embodiments, the intracellular signaling domainincludes DAP10/CD28 type signaling chains. In some embodiments, theintracellular signaling domain is not covalently attached to themembrane bound CAR, but is instead diffused in the cytoplasm.

Intracellular signaling domains suitable for use in a subject CAR of thepresent invention include immunoreceptor tyrosine-based activation motif(ITAM)-containing intracellular signaling polypeptides. In someembodiments, an ITAM motif is repeated twice in an intracellularsignaling domain, where the first and second instances of the ITAM motifare separated from one another by 6 to 8 amino acids. In one embodiment,the intracellular signaling domain of a subject CAR comprises 3 ITAMmotifs.

In some embodiments, intracellular signaling domains includes thesignaling domains of human immunoglobulin receptors that containimmunoreceptor tyrosine based activation motifs (ITAMs) such as, but notlimited to, FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5(see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).

A suitable intracellular signaling domain can be an ITAMmotif-containing portion that is derived from a polypeptide thatcontains an ITAM motif. For example, a suitable intracellular signalingdomain can be an ITAM motif-containing domain from any ITAMmotif-containing protein. Thus, a suitable intracellular signalingdomain need not contain the entire sequence of the entire protein fromwhich it is derived. Examples of suitable ITAM motif-containingpolypeptides include, but are not limited to: DAP12, FCER1G (Fc epsilonreceptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associatedprotein alpha chain).

In one embodiment, the intracellular signaling domain is derived fromDAP12 (also known as TYROBP; TYRO protein tyrosine kinase bindingprotein; KARAP; PLOSL; DNAX-activation protein 12; KAR-associatedprotein; TYRO protein tyrosine kinase-binding protein; killer activatingreceptor associated protein; killer-activating receptor-associatedprotein; etc.). In one embodiment, the intracellular signaling domain isderived from FCER1G (also known as FCRG; Fc epsilon receptor I gammachain; Fc receptor gamma-chain; fc-epsilon RI-gamma; fcRgamma; fceRlgamma; high affinity immunoglobulin epsilon receptor subunit gamma;immunoglobulin E receptor, high affinity, gamma chain; etc.). In oneembodiment, the intracellular signaling domain is derived from T-cellsurface glycoprotein CD3 delta chain (also known as CD3D; CD3-DELTA;T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, deltapolypeptide (TiT3 complex); OKT3, delta chain; T-cell receptor T3 deltachain; T-cell surface glycoprotein CD3 delta chain; etc.). In oneembodiment, the intracellular signaling domain is derived from T-cellsurface glycoprotein CD3 epsilon chain (also known as CD3e, T-cellsurface antigen T3/Leu-4 epsilon chain, T-cell surface glycoprotein CD3epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.). In one embodiment,the intracellular signaling domain is derived from T-cell surfaceglycoprotein CD3 gamma chain (also known as CD3G, T-cell receptor T3gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). Inone embodiment, the intracellular signaling domain is derived fromT-cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T-cellreceptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.).In one embodiment, the intracellular signaling domain is derived fromCD79A (also known as B-cell antigen receptor complex-associated proteinalpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1membrane glycoprotein; ig-alpha; membrane-boundimmunoglobulin-associated protein; surface IgM-associated protein;etc.). In one embodiment, an intracellular signaling domain suitable foruse in an FN3 CAR of the present disclosure includes a DAP10/CD28 typesignaling chain. In one embodiment, an intracellular signaling domainsuitable for use in an FN3 CAR of the present disclosure includes aZAP70 polypeptide. In some embodiments, the intracellular signalingdomain includes a cytoplasmic signaling domain of TCR zeta, FcR gamma,FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, orCD66d. In one embodiment, the intracellular signaling domain in the CARincludes a cytoplasmic signaling domain of human CD3 zeta.

While usually the entire intracellular signaling domain can be employed,in many cases it is not necessary to use the entire chain. To the extentthat a truncated portion of the intracellular signaling domain is used,such truncated portion may be used in place of the intact chain as longas it transduces the effector function signal. The intracellularsignaling domain includes any truncated portion of the intracellularsignaling domain sufficient to transduce the effector function signal.

The intracellular signaling domains described herein can be combinedwith any of the antigen binding domains described herein, any of thetransmembrane domains described herein, or any of the other domainsdescribed herein that may be included in the CAR.

D. Nucleic Acids and Expression Vectors

The present disclosure provides a nucleic acid encoding an exogenous TCRand/or CAR. In one embodiment, a nucleic acid of the present disclosurecomprises a nucleic acid sequence encoding an exogenous TCR (e.g., anNY-ESO-1 TCR). In one embodiment, a nucleic acid of the presentdisclosure comprises a nucleic acid sequence encoding an exogenous CAR(e.g., a PSCA CAR).

In some embodiments, a nucleic acid of the present disclosure isprovided for the production of a TCR and/or CAR as described herein,e.g., in a mammalian cell. In some embodiments, a nucleic acid of thepresent disclosure provides for amplification of the TCR- orCAR-encoding nucleic acid.

As described herein, a TCR of the present disclosure comprises a TCRalpha chain and a TCR beta chain. Accordingly, the present disclosureprovides a nucleic acid encoding a TCR alpha chain, and a nucleic acidencoding a TCR beta chain. In some embodiments, the nucleic acidencoding a TCR alpha chain is separate from the nucleic acid encoding aTCR beta chain. In an exemplary embodiment, the nucleic acid encoding aTCR alpha chain, and the nucleic acid encoding a TCR beta chain, resideswithin the same nucleic acid.

In some embodiments, a nucleic acid of the present disclosure comprisesa nucleic acid comprising a TCR alpha chain coding sequence and a TCRbeta chain coding sequence. In some embodiments, a nucleic acid of thepresent disclosure comprises a nucleic acid comprising a TCR alpha chaincoding sequence and a TCR beta chain coding sequence that is separatedby a linker. A linker for use in the present disclosure allows formultiple proteins to be encoded by the same nucleic acid sequence (e.g.,a multicistronic or bicistronic sequence), which are translated as apolyprotein that is dissociated into separate protein components. Forexample, a linker for use in a nucleic acid of the present disclosurecomprising a TCR alpha chain coding sequence and a TCR beta chain codingsequence, allows for the TCR alpha chain and TCR beta chain to betranslated as a polyprotein that is dissociated into separate TCR alphachain and TCR beta chain components.

In some embodiments, the linker comprises a nucleic acid sequence thatencodes for an internal ribosome entry site (IRES). As used herein, “aninternal ribosome entry site” or “IRES” refers to an element thatpromotes direct internal ribosome entry to the initiation codon, such asATG, of a protein coding region, thereby leading to cap-independenttranslation of the gene. Various internal ribosome entry sites are knownto those of skill in the art, including, without limitation, IRESobtainable from viral or cellular mRNA sources, e.g., immunogloublinheavy-chain binding protein (BiP); vascular endothelial growth factor(VEGF); fibroblast growth factor 2; insulin-like growth factor;translational initiation factor eIF4G; yeast transcription factors TFIIDand HAP4; and IRES obtainable from, e.g., cardiovirus, rhinovirus,aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloneymurine leukemia virus (MoMLV). Those of skill in the art would be ableto select the appropriate IRES for use in the present invention.

In some embodiments, the linker comprises a nucleic acid sequence thatencodes for a self-cleaving peptide. As used herein, a “self-cleavingpeptide” or “2A peptide” refers to an oligopeptide that allow multipleproteins to be encoded as polyproteins, which dissociate into componentproteins upon translation. Use of the term “self-cleaving” is notintended to imply a proteolytic cleavage reaction. Various self-cleavingor 2A peptides are known to those of skill in the art, including,without limitation, those found in members of the Picornaviridae virusfamily, e.g., foot-and-mouth disease virus (FMDV), equine rhinitis Avirus (ERAV0, Thosea asigna virus (TaV), and porcine tescho virus-1(PTV-1); and carioviruses such as Theilovirus and encephalomyocarditisviruses. 2A peptides derived from FMDV, ERAV, PTV-1, and TaV arereferred to herein as “F2A,” “E2A,” “P2A,” and “T2A,” respectively.Those of skill in the art would be able to select the appropriateself-cleaving peptide for use in the present invention.

In some embodiments, a linker further comprises a nucleic acid sequencethat encodes a furin cleavage site. Furin is a ubiquitously expressedprotease that resides in the trans-golgi and processes proteinprecursors before their secretion. Furin cleaves at the COOH— terminusof its consensus recognition sequence. Various furin consensusrecognition sequences (or “furin cleavage sites”) are known to those ofskill in the art, including, without limitation, Arg-X1-Lys-Arg (SEQ IDNO:26) or Arg-X1-Arg-Arg (SEQ ID NO:27), X2-Arg-X1-X3-Arg (SEQ ID NO:28)and Arg-X1-X1-Arg (SEQ ID NO:29), such as an Arg-Gln-Lys-Arg (SEQ IDNO:30), where X1 is any naturally occurring amino acid, X2 is Lys orArg, and X3 is Lys or Arg. Those of skill in the art would be able toselect the appropriate Furin cleavage site for use in the presentinvention.

In some embodiments, the linker comprises a nucleic acid sequenceencoding a combination of a Furin cleavage site and a 2A peptide.Examples include, without limitation, a linker comprising a nucleic acidsequence encoding Furin and F2A, a linker comprising a nucleic acidsequence encoding Furin and E2A, a linker comprising a nucleic acidsequence encoding Furin and P2A, a linker comprising a nucleic acidsequence encoding Furin and T2A. Those of skill in the art would be ableto select the appropriate combination for use in the present invention.In such embodiments, the linker may further comprise a spacer sequencebetween the Furin and 2A peptide. Various spacer sequences are known inthe art, including, without limitation, glycine serine (GS) spacers suchas (GS)n, (GSGGS)n (SEQ ID NO:2) and (GGGS)n (SEQ ID NO:3), where nrepresents an integer of at least 1. Exemplary spacer sequences cancomprise amino acid sequences including, without limitation, GGSG (SEQID NO:5), GGSGG (SEQ ID NO:6), GSGSG (SEQ ID NO:7), GSGGG (SEQ ID NO:8),GGGSG (SEQ ID NO:9), GSSSG (SEQ ID NO:10), and the like. Those of skillin the art would be able to select the appropriate spacer sequence foruse in the present invention.

In some embodiments, a nucleic acid of the present disclosure may beoperably linked to a transcriptional control element, e.g., a promoter,and enhancer, etc. Suitable promoter and enhancer elements are known tothose of skill in the art.

For expression in a bacterial cell, suitable promoters include, but arenot limited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. Forexpression in a eukaryotic cell, suitable promoters include, but are notlimited to, light and/or heavy chain immunoglobulin gene promoter andenhancer elements; cytomegalovirus immediate early promoter; herpessimplex virus thymidine kinase promoter; early and late SV40 promoters;promoter present in long terminal repeats from a retrovirus; mousemetallothionein-I promoter; and various art-known tissue specificpromoters. Suitable reversible promoters, including reversible induciblepromoters are known in the art. Such reversible promoters may beisolated and derived from many organisms, e.g., eukaryotes andprokaryotes. Modification of reversible promoters derived from a firstorganism for use in a second organism, e.g., a first prokaryote and asecond a eukaryote, a first eukaryote and a second a prokaryote, etc.,is well known in the art. Such reversible promoters, and systems basedon such reversible promoters but also comprising additional controlproteins, include, but are not limited to, alcohol regulated promoters(e.g., alcohol dehydrogenase I (alcA) gene promoter, promotersresponsive to alcohol transactivator proteins (A1cR), etc.),tetracycline regulated promoters, (e.g., promoter systems includingTetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g.,rat glucocorticoid receptor promoter systems, human estrogen receptorpromoter systems, retinoid promoter systems, thyroid promoter systems,ecdysone promoter systems, mifepristone promoter systems, etc.), metalregulated promoters (e.g., metallothionein promoter systems, etc.),pathogenesis-related regulated promoters (e.g., salicylic acid regulatedpromoters, ethylene regulated promoters, benzothiadiazole regulatedpromoters, etc.), temperature regulated promoters (e.g., heat shockinducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter,etc.), light regulated promoters, synthetic inducible promoters, and thelike.

In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4cell-specific promoter, a neutrophil-specific promoter, or anNK-specific promoter. For example, a CD4 gene promoter can be used; see,e.g., Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; andMarodon et al. (2003) Blood 101:3416. As another example, a CD8 genepromoter can be used. NK cell-specific expression can be achieved by useof an NcrI (p46) promoter; see, e.g., Eckelhart et al. Blood (2011)117:1565.

For expression in a yeast cell, a suitable promoter is a constitutivepromoter such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, aPYK1 promoter and the like; or a regulatable promoter such as a GAL1promoter, a GAL10 promoter, an ADH2 promoter, a PHOS promoter, a CUP1promoter, a GALT promoter, a MET25 promoter, a MET3 promoter, a CYC1promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDHpromoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2promoter, an ENO promoter, a TP1 promoter, and AOX1 (e.g., for use inPichia). Selection of the appropriate vector and promoter is well withinthe level of ordinary skill in the art. Suitable promoters for use inprokaryotic host cells include, but are not limited to, a bacteriophageT7 RNA polymerase promoter; a trp promoter; a lac operon promoter; ahybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybridpromoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tacpromoter, and the like; an araBAD promoter; in vivo regulated promoters,such as an ssaG promoter or a related promoter (see, e.g., U.S. PatentPublication No. 20040131637), a pagC promoter (Pulkkinen and Miller, J.Bacteriol. (1991) 173(1): 86-93; Alpuche-Aranda et al., Proc. Natl.Acad. Sci. USA (1992) 89(21): 10079-83), a nirB promoter (Harborne etal. Mol. Micro. (1992) 6:2805-2813), and the like (see, e.g., Dunstan etal., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004)22:3243-3255; and Chatfield et al., Biotechnol. (1992) 10:888-892); asigma70 promoter, e.g., a consensus sigma70 promoter (see, e.g., GenBankAccession Nos. AX798980, AX798961, and AX798183); a stationary phasepromoter, e.g., a dps promoter, an spy promoter, and the like; apromoter derived from the pathogenicity island SPI-2 (see, e.g.,WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al., Infect.Immun. (2002) 70:1087-1096); an rpsM promoter (see, e.g., Valdivia andFalkow Mol. Microbiol. (1996). 22:367); a tet promoter (see, e.g.,Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U.(eds), Topics in Molecular and Structural Biology, Protein—Nucleic AcidInteraction. Macmillan, London, UK, Vol. 10, pp. 143-162); an SP6promoter (see, e.g., Melton et al., Nucl. Acids Res. (1984) 12:7035);and the like. Suitable strong promoters for use in prokaryotes such asEscherichia coli include, but are not limited to Trc, Tac, T5, T7, andPLambda. Non-limiting examples of operators for use in bacterial hostcells include a lactose promoter operator (LacI repressor proteinchanges conformation when contacted with lactose, thereby preventing theLad repressor protein from binding to the operator), a tryptophanpromoter operator (when complexed with tryptophan, TrpR repressorprotein has a conformation that binds the operator; in the absence oftryptophan, the TrpR repressor protein has a conformation that does notbind to the operator), and a tac promoter operator (see, e.g., deBoer etal., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:21-25).

Other examples of suitable promoters include the immediate earlycytomegalovirus (CMV) promoter sequence. This promoter sequence is astrong constitutive promoter sequence capable of driving high levels ofexpression of any polynucleotide sequence operatively linked thereto.Other constitutive promoter sequences may also be used, including, butnot limited to a simian virus 40 (SV40) early promoter, a mouse mammarytumor virus (MMTV) or human immunodeficiency virus (HIV) long terminalrepeat (LTR) promoter, a MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcomavirus promoter, the EF-1 alpha promoter, as well as human gene promoterssuch as, but not limited to, an actin promoter, a myosin promoter, ahemoglobin promoter, and a creatine kinase promoter. Further, theinvention should not be limited to the use of constitutive promoters.Inducible promoters are also contemplated as part of the invention. Theuse of an inducible promoter provides a molecular switch capable ofturning on expression of the polynucleotide sequence which it isoperatively linked when such expression is desired, or turning off theexpression when expression is not desired. Examples of induciblepromoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracyclinepromoter.

In some embodiments, the locus or construct or transgene containing thesuitable promoter is irreversibly switched through the induction of aninducible system. Suitable systems for induction of an irreversibleswitch are well known in the art, e.g., induction of an irreversibleswitch may make use of a Cre-lox-mediated recombination (see, e.g.,Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99,the disclosure of which is incorporated herein by reference). Anysuitable combination of recombinase, endonuclease, ligase, recombinationsites, etc. known to the art may be used in generating an irreversiblyswitchable promoter. Methods, mechanisms, and requirements forperforming site-specific recombination, described elsewhere herein, finduse in generating irreversibly switched promoters and are well known inthe art, see, e.g., Grindley et al. Annual Review of Biochemistry (2006)567-605; and Tropp, Molecular Biology (2012) (Jones & BartlettPublishers, Sudbury, Mass.), the disclosures of which are incorporatedherein by reference.

In some embodiments, a nucleic acid of the present disclosure furthercomprises a nucleic acid sequence encoding a TCR and/or CAR inducibleexpression cassette. In one embodiment, the TCR and/or CAR inducibleexpression cassette is for the production of a transgenic polypeptideproduct that is released upon TCR and/or CAR signaling. See, e.g.,Chmielewski and Abken, Expert Opin. Biol. Ther. (2015) 15(8): 1145-1154;and Abken, Immunotherapy (2015) 7(5): 535-544. In some embodiments, anucleic acid of the present disclosure further comprises a nucleic acidsequence encoding a cytokine operably linked to a T-cell activationresponsive promoter. In some embodiments, the cytokine operably linkedto a T-cell activation responsive promoter is present on a separatenucleic acid sequence. In one embodiment, the cytokine is IL-12.

A nucleic acid of the present disclosure may be present within anexpression vector and/or a cloning vector. An expression vector caninclude a selectable marker, an origin of replication, and otherfeatures that provide for replication and/or maintenance of the vector.Suitable expression vectors include, e.g., plasmids, viral vectors, andthe like. Large numbers of suitable vectors and promoters are known tothose of skill in the art; many are commercially available forgenerating a subject recombinant construct. The following vectors areprovided by way of example, and should not be construed in anyway aslimiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS,pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA);pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala,Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene)pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Suitable expression vectorsinclude, but are not limited to, viral vectors (e.g. viral vectors basedon vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest.Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther.(1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995)92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655); adeno-associated virus (see, e.g., Ali et al., Hum. GeneTher. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA(1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci.(1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690,Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum.Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93/09239, Samulski etal., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus(see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94:10319-23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); aretroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus,and vectors derived from retroviruses such as Rous Sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, human immunodeficiency virus,myeloproliferative sarcoma virus, and mammary tumor virus); and thelike.

Additional expression vectors suitable for use are, e.g., withoutlimitation, a lentivirus vector, a gamma retrovirus vector, a foamyvirus vector, an adeno-associated virus vector, an adenovirus vector, apox virus vector, a herpes virus vector, an engineered hybrid virusvector, a transposon mediated vector, and the like. Viral vectortechnology is well known in the art and is described, for example, inSambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes1-4, Cold Spring Harbor Press, NY), and in other virology and molecularbiology manuals. Viruses, which are useful as vectors include, but arenot limited to, retroviruses, adenoviruses, adeno-associated viruses,herpes viruses, and lentiviruses.

In general, a suitable vector contains an origin of replicationfunctional in at least one organism, a promoter sequence, convenientrestriction endonuclease sites, and one or more selectable markers,(e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

In some embodiments, an expression vector (e.g., a lentiviral vector)may be used to introduce the TCR and/or CAR into an immune cell orprecursor thereof (e.g., a T cell). Accordingly, an expression vector(e.g., a lentiviral vector) of the present invention may comprise anucleic acid encoding for a TCR and/or CAR. In some embodiments, theexpression vector (e.g., lentiviral vector) will comprise additionalelements that will aid in the functional expression of the TCR and/orCAR encoded therein. In some embodiments, an expression vectorcomprising a nucleic acid encoding for a TCR and/or CAR furthercomprises a mammalian promoter. In one embodiment, the vector furthercomprises an elongation-factor-1-alpha promoter (EF-1α promoter). Use ofan EF-1α promoter may increase the efficiency in expression ofdownstream transgenes (e.g., a TCR and/or CAR encoding nucleic acidsequence). Physiologic promoters (e.g., an EF-1α promoter) may be lesslikely to induce integration mediated genotoxicity, and may abrogate theability of the retroviral vector to transform stem cells. Otherphysiological promoters suitable for use in a vector (e.g., lentiviralvector) are known to those of skill in the art and may be incorporatedinto a vector of the present invention. In some embodiments, the vector(e.g., lentiviral vector) further comprises a non-requisite cis actingsequence that may improve titers and gene expression. One non-limitingexample of a non-requisite cis acting sequence is the central polypurinetract and central termination sequence (cPPT/CTS) which is important forefficient reverse transcription and nuclear import. Other non-requisitecis acting sequences are known to those of skill in the art and may beincorporated into a vector (e.g., lentiviral vector) of the presentinvention. In some embodiments, the vector further comprises aposttranscriptional regulatory element. Posttranscriptional regulatoryelements may improve RNA translation, improve transgene expression andstabilize RNA transcripts. One example of a posttranscriptionalregulatory element is the woodchuck hepatitis virus posttranscriptionalregulatory element (WPRE). Accordingly, in some embodiments a vector forthe present invention further comprises a WPRE sequence. Variousposttranscriptional regulator elements are known to those of skill inthe art and may be incorporated into a vector (e.g., lentiviral vector)of the present invention. A vector of the present invention may furthercomprise additional elements such as a rev response element (RRE) forRNA transport, packaging sequences, and 5′ and 3′ long terminal repeats(LTRs). The term “long terminal repeat” or “LTR” refers to domains ofbase pairs located at the ends of retroviral DNAs which comprise U3, Rand U5 regions. LTRs generally provide functions required for theexpression of retroviral genes (e.g., promotion, initiation andpolyadenylation of gene transcripts) and to viral replication. In oneembodiment, a vector (e.g., lentiviral vector) of the present inventionincludes a 3′ U3 deleted LTR. Accordingly, a vector (e.g., lentiviralvector) of the present invention may comprise any combination of theelements described herein to enhance the efficiency of functionalexpression of transgenes. For example, a vector (e.g., lentiviralvector) of the present invention may comprise a WPRE sequence, cPPTsequence, RRE sequence, 5′LTR, 3′ U3 deleted LTR′ in addition to anucleic acid encoding for a TCR and/or CAR.

Vectors of the present invention may be self-inactivating vectors. Asused herein, the term “self-inactivating vector” refers to vectors inwhich the 3′ LTR enhancer promoter region (U3 region) has been modified(e.g., by deletion or substitution). A self-inactivating vector mayprevent viral transcription beyond the first round of viral replication.Consequently, a self-inactivating vector may be capable of infecting andthen integrating into a host genome (e.g., a mammalian genome) onlyonce, and cannot be passed further. Accordingly, self-inactivatingvectors may greatly reduce the risk of creating a replication-competentvirus.

In some embodiments, a nucleic acid of the present invention may be RNA,e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNAare known to those of skill in the art; any known method can be used tosynthesize RNA comprising a sequence encoding a TCR and/or CAR of thepresent disclosure. Methods for introducing RNA into a host cell areknown in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053.Introducing RNA comprising a nucleotide sequence encoding a TCR and/orCAR of the present disclosure into a host cell can be carried out invitro, ex vivo or in vivo. For example, a host cell (e.g., an NK cell, acytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivowith RNA comprising a nucleotide sequence encoding a TCR and/or CAR ofthe present disclosure.

In order to assess the expression of a polypeptide or portions thereof,the expression vector to be introduced into a cell may also containeither a selectable marker gene or a reporter gene, or both, tofacilitate identification and selection of expressing cells from thepopulation of cells sought to be transfected or infected through viralvectors. In some embodiments, the selectable marker may be carried on aseparate piece of DNA and used in a co-transfection procedure. Bothselectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Usefulselectable markers include, without limitation, antibiotic-resistancegenes.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Ingeneral, a reporter gene is a gene that is not present in or expressedby the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g.,enzymatic activity. Expression of the reporter gene is assessed at asuitable time after the DNA has been introduced into the recipientcells. Suitable reporter genes may include, without limitation, genesencoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescentprotein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

E. Modified Immune Cells

The present invention provides a modified immune cell or precursorthereof (e.g., a T cell) comprising an exogenous TCR and/or CAR asdescribed herein. Accordingly, such modified cells possess thespecificity directed by the TCR and/or CAR that is expressed therein.For example, a modified cell of the present disclosure comprising aNY-ESO-1 TCR possesses specificity for NY-ESO-1 on a target cell.

Gene Edited Immune Cells

The present disclosure provides gene edited modified cells. In someembodiments, a modified cell (e.g., a modified cell comprising anexogenous TCR and/or CAR) of the present disclosure is geneticallyedited to disrupt the expression of one or more endogenously expressedgenes. In some embodiments, the gene-edited immune cells (e.g., T cells)have a reduction, deletion, elimination, knockout or disruption inexpression of one or more endogenously expressed genes

In some embodiments, the modified cell of the present disclosure isgenetically edited to disrupt the expression of one or more of anendogenous gene selected from the group consisting of Apolipoprotein A2(APOA2), 5-Azacytidine Induced 2 (AZI2), BTB Domain And CNC Homolog 2(Bach2), C10orf129 (also known as Acyl-CoA Synthetase Medium ChainFamily Member 6; ACSM6), C1orf141, C1orf64 (also known as SteroidReceptor Associated And Regulated Protein; SRARP), C-C Motif ChemokineLigand 7 (CCL7), Cyclin I Family Member 2 (CCNI2), Chloride ChannelAccessory 1 (CLCA1), Chloride Nucleotide-Sensitive Channel 1A (CLNS1A),Clock Circadian Regulator (CLOCK), Cysteine Rich Transmembrane ModuleContaining 1 (CYSTM1), Defensin Alpha 4 (DEFA4), Family With SequenceSimilarity 124 Member A (FAM124A), FAM86A (also known as EukaryoticElongation Factor 2 Lysine Methyltransferase; EEF2KMT), PolypeptideN-Acetylgalactosaminyltransferase 2 (GALNT2), Glyoxalase DomainContaining 5 (GLODS), G Protein-Coupled Receptor 35 (GPR35),Hypoxia-Regulated Factor-1 (HRF1), Histidine Rich Glycoprotein (HRG),hsa-mir1273d, hsa-mir-6505, Kinesin Family Member 27 (KIF27), KruppelLike Factor 4 (K1f4), Myosin Binding Protein H (MYBPH), NDC,NADH:Ubiquinone Oxidoreductase Subunit S4 (NDUFS4), PAXIP1 AssociatedGlutamate Rich Protein 1 (PAGR1), Parvin Gamma (PARVG),Phosphomevalonate Kinase (PMVK), Protein Kinase CAMP-Activated CatalyticSubunit Beta (PRKACB), Pre-MRNA Processing Factor 39 (PRPF39), PregnancySpecific Beta-1-Glycoprotein 5 (PSGS), Prostaglandin 12 Receptor(PTGIR), Poliovirus Receptor-Related 3 (PVRL3), SIX Homeobox 2 (SIX2),Transmembrane Protein 249 (TMEM249), Transmembrane Protein 48 (TMEM48),Tetratricopeptide Repeat Domain 27 (TTC27), Ubiquitin Specific Peptidase27, X-Linked (USP27X), WD Repeat-Containing Protein 85 (WDR85), YY1Associated Protein 1 (YY1AP), and Zinc And Ring Finger 2 (ZNRF2).

Immunotherapies using CAR (chimeric antigen receptors) T cells and TCRredirected T cells have shown various efficacies in the treatment ofcancer patients. One of the major problems limiting their effects isthat T cells are exhausted after persistent stimulation by tumor cells.Exhausted T cells have reduced effector functions such as production ofcytokines and cytotoxicity against tumor cells, and they express higherlevels of checkpoint inhibitory molecules, such as PD-1 and CTLA-4. PD-1and CTLA-4 antibodies have been used clinically to treat multiple typesof cancers. However, the majority of patients do not benefitsignificantly from these therapies. It has been shown that thegenome-wide epigenetic landscape of exhausted T cells is different fromthat of effector T cells and memory T cells, and these exhausted T cellscannot be remodeled/reinvigorated by, e.g., PD-L1 blockade (see, Paukenet al. (2016) Science, 354(6316):1160-1165). Without being bound to anytheory, the altered epigenetic landscape may be limiting the exhausted Tcell from being fully reinvigorated by checkpoint antibodies.

In certain embodiments, the modified cell of the present disclosure isgenetically edited to disrupt the expression of a transcriptionalmodulator. As described elsewhere herein, disruption of atranscriptional modulator (e.g., a transcription factor or an epigeneticregulator) is shown by the present disclosure to enhance immune cell(e.g., T cell) function. Without being bound to any theory, disruptingthe expression of a transcriptional modulator (e.g., a transcriptionfactor or an epigenetic regulator) may result in reduced expression ofgenes involved in negatively regulating immune cell function (e.g., Tcell survival) and/or enhanced expression of genes involved inpositively regulating immune cell function (e.g., pro-survival factors),thus increasing efficacy of the gene edited immune cells.

Accordingly, a modified cell of the present disclosure with disruptedexpression of a transcriptional modulator (e.g., a modified T cell withdisrupted expression of a transcription factor or an epigeneticregulator), may be resistant to exhaustion, and in some embodiments, mayadditionally be reinvigorated by checkpoint antibodies (e.g., anti-PD-1,anti-CTLA-4, anti-PDL1 antibodies).

For example, the present disclosure identifies PAGR1, Klf4, and SIX2 asgenes that when expression is disrupted, enhances immune cell (e.g., Tcell) function. In an exemplary embodiment, a modified cell of thepresent disclosure is genetically edited to disrupt the expression ofPAGR1. In an exemplary embodiment, a modified cell of the presentdisclosure is genetically edited to disrupt the expression of Klf4. Inan exemplary embodiment, a modified cell of the present disclosure isgenetically edited to disrupt the expression of SIX2. In someembodiments, a modified cell of the present disclosure is geneticallyedited to disrupt the expression of one or more genes selected from thegroup consisting of PAGR1, Klf4, and SIX2.

Where disruption of the expression of a transcriptional modulator (e.g.,a transcription factor or an epigenetic regulator) results in thedownregulation of the expression of one or more genes that actdownstream of the transcriptional modulator, a modified cell of thepresent invention can be genetically edited to disrupt the expression ofone or more of the downstream acting genes. For example, Klf4 and SIX2are transcription factors that regulate the expression of one or moredownstream genes that are known in the art. Accordingly, the presentinvention provides a modified cell genetically edited to disrupt theexpression of one or more of the genes that are regulated by Klf4 and/orSIX2. In another example, PAGR1 is a known component of the epigeneticregulator histone-lysine-N-methyltransferase 2D (KMT2D). As describedelsewhere herein, disruption of PAGR1 results in the reduced expressionof one or more of AT-Rich Interaction Domain 1A (ARID1A), AT-RichInteraction Domain 3B (ARID3B), Additional Sex Combs Like 1 (ASXL1), DNAMethyltransferase 3 Alpha (DNMT3A), Dual Specificity Phosphatase 1(DUSP1), Mitogen-Activated Protein Kinase Kinase Kinase 8 (MAP3K8), PAXInteracting Protein 1 (PAXIP1), Protein Arginine Methyltransferase 1(PRMT1), Suppressor Of Cytokine Signaling 3 (SOCS3), and/or TNF AlphaInduced Protein 3 (TNFAIP3). Accordingly, the present invention providesa modified cell genetically edited to disrupt the expression of one ormore of the genes selected from the group consisting of ARID1A, ARID3B,ASXL1, DNMT3A, DUSP1, MAP3K8, PAXIP1, PRMT1, SOCS3, and TNFAIP3.

In other embodiments, the modified cell of the present disclosure isgenetically edited to disrupt the expression of endogenous PDCD1 geneproducts (Programmed Death 1 receptor; PD-1). Disrupting the expressionof endogenous PD-1 may create “checkpoint” resistant modified cells,resulting in increased tumor control. Checkpoint resistant modifiedcells may also be created by disrupting the expression of, for example,without limitation, the Adenosine A2A receptor (A2AR), B7-H3 (CD276),B7-H4 (VTCN1), the B and T Lymphocyte Attenuator protein (BTLA/CD272),CD96, the Cytotoxic T-Lymphocyte Associated protein 4 (CTLA-4/CD152),Indoleamine 2,3-dioxygenase (IDO), the Killer-cell Immunoglobulin-likeReceptor (KIR), the Lymphocyte Activation Gene-3 (LAG3), the T cellimmunoreceptor with Ig and ITIM domains (TIGIT), T-cell Immunoglobulindomain and Mucin domain 3 (TIM-3), or the V-domain Ig suppressor of Tcell activation (VISTA).

Accordingly, the modified cell of the present invention is geneticallyedited to disrupt the expression of any of the endogenous genesdescribed herein. Accordingly, in some embodiments, a modified cell(e.g., a modified cell comprising an exogenous TCR and/or CAR) of thepresent invention is genetically edited to disrupt the expression of oneor more of the endogenous genes described herein.

Various gene editing technologies are known to those skilled in the art.Gene editing technologies include, without limitation, homingendonucleases, zinc-finger nucleases (ZFNs), transcriptionactivator-like effector (TALE) nucleases (TALENs), and clusteredregularly interspaced short palindromic repeats(CRISPR)-CRISPR-associated protein 9 (Cas9). Homing endonucleasesgenerally cleave their DNA substrates as dimers, and do not havedistinct binding and cleavage domains. ZFNs recognize target sites thatconsist of two zinc-finger binding sites that flank a 5- to 7-base pair(bp) spacer sequence recognized by the FokI cleavage domain. TALENsrecognize target sites that consist of two TALE DNA-binding sites thatflank a 12- to 20-bp spacer sequence recognized by the FokI cleavagedomain. The Cas9 nuclease is targeted to DNA sequences complementary tothe targeting sequence within the single guide RNA (gRNA) locatedimmediately upstream of a compatible protospacer adjacent motif (PAM).Accordingly, one of skill in the art would be able to select theappropriate gene editing technology for the present invention.

In some aspects, the disruption is carried out by gene editing using anRNA-guided nuclease such as a CRISPR-Cas system, such as CRISPR-Cas9system, specific for the gene (e.g., PAGR1, Klf4, SIX2) being disrupted.In some embodiments, an agent containing a Cas9 and a guide RNA (gRNA)containing a targeting domain, which targets a region of the geneticlocus, is introduced into the cell. In some embodiments, the agent is orcomprises a ribonucleoprotein (RNP) complex of a Cas9 polypeptide and agRNA (Cas9/gRNA RNP). In some embodiments, the introduction includescontacting the agent or portion thereof with the cells in vitro, whichcan include cultivating or incubating the cell and agent for up to 24,36 or 48 hours or 3, 4, 5, 6, 7, or 8 days. In some embodiments, theintroduction further can include effecting delivery of the agent intothe cells. In various embodiments, the methods, compositions and cellsaccording to the present disclosure utilize direct delivery ofribonucleoprotein (RNP) complexes of Cas9 and gRNA to cells, for exampleby electroporation. In some embodiments, the RNP complexes include agRNA that has been modified to include a 3′ poly-A tail and a 5′Anti-Reverse Cap Analog (ARCA) cap.

The CRISPR/Cas9 system is a facile and efficient system for inducingtargeted genetic alterations. Target recognition by the Cas9 proteinrequires a ‘seed’ sequence within the guide RNA (gRNA) and a conserveddi-nucleotide containing protospacer adjacent motif (PAM) sequenceupstream of the gRNA-binding region. The CRISPR/Cas9 system can therebybe engineered to cleave virtually any DNA sequence by redesigning thegRNA in cell lines (such as 293T cells), primary cells, and TCR T cells.The CRISPR/Cas9 system can simultaneously target multiple genomic lociby co-expressing a single Cas9 protein with two or more gRNAs, makingthis system suited for multiple gene editing or synergistic activationof target genes.

The Cas9 protein and guide RNA form a complex that identifies andcleaves target sequences. Cas9 is comprised of six domains: REC I, RECII, Bridge Helix, PAM interacting, HNH, and RuvC. The REC I domain bindsthe guide RNA, while the Bridge helix binds to target DNA. The HNH andRuvC domains are nuclease domains. Guide RNA is engineered to have a 5′end that is complementary to the target DNA sequence. Upon binding ofthe guide RNA to the Cas9 protein, a conformational change occursactivating the protein. Once activated, Cas9 searches for target DNA bybinding to sequences that match its protospacer adjacent motif (PAM)sequence. A PAM is a two or three nucleotide base sequence within onenucleotide downstream of the region complementary to the guide RNA. Inone non-limiting example, the PAM sequence is 5′-NGG-3′. When the Cas9protein finds its target sequence with the appropriate PAM, it melts thebases upstream of the PAM and pairs them with the complementary regionon the guide RNA. Then the RuvC and HNH nuclease domains cut the targetDNA after the third nucleotide base upstream of the PAM.

One non-limiting example of a CRISPR/Cas system used to inhibit geneexpression, CRISPRi, is described in U.S. Patent Appl. Publ. No.US20140068797. CRISPRi induces permanent gene disruption that utilizesthe RNA-guided Cas9 endonuclease to introduce DNA double stranded breakswhich trigger error-prone repair pathways to result in frame shiftmutations. A catalytically dead Cas9 lacks endonuclease activity. Whencoexpressed with a guide RNA, a DNA recognition complex is generatedthat specifically interferes with transcriptional elongation, RNApolymerase binding, or transcription factor binding. This CRISPRi systemefficiently represses expression of targeted genes.

CRISPR/Cas gene disruption occurs when a guide nucleic acid sequencespecific for a target gene and a Cas endonuclease are introduced into acell and form a complex that enables the Cas endonuclease to introduce adouble strand break at the target gene. In certain embodiments, theCRISPR/Cas system comprises an expression vector, such as, but notlimited to, a pAd5F35-CRISPR vector. In other embodiments, the Casexpression vector induces expression of Cas9 endonuclease. Otherendonucleases may also be used, including but not limited to, Cas12a(Cpf1), T7, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10,Csm2, Cmr5, Fok1, other nucleases known in the art, and any combinationsthereof.

In certain embodiments, inducing the Cas expression vector comprisesexposing the cell to an agent that activates an inducible promoter inthe Cas expression vector. In such embodiments, the Cas expressionvector includes an inducible promoter, such as one that is inducible byexposure to an antibiotic (e.g., by tetracycline or a derivative oftetracycline, for example doxycycline). Other inducible promoters knownby those of skill in the art can also be used. The inducing agent can bea selective condition (e.g., exposure to an agent, for example anantibiotic) that results in induction of the inducible promoter. Thisresults in expression of the Cas expression vector.

As used herein, the term “guide RNA” or “gRNA” refer to any nucleic acidthat promotes the specific association (or “targeting”) of an RNA-guidednuclease such as a Cas9 to a target sequence (e.g., a genomic orepisomal sequence) in a cell.

As used herein, a “modular” or “dual RNA” guide comprises more than one,and typically two, separate RNA molecules, such as a CRISPR RNA (crRNA)and a trans-activating crRNA (tracrRNA), which are usually associatedwith one another, for example by duplexing. gRNAs and their componentparts are described throughout the literature (see, e.g., Briner et al.Mol. Cell, 56(2), 333-339 (2014), which is incorporated by reference).

As used herein, a “unimolecular gRNA,” “chimeric gRNA,” or “single guideRNA (sgRNA)” comprises a single RNA molecule. The sgRNA may be a crRNAand tracrRNA linked together. For example, the 3′ end of the crRNA maybe linked to the 5′ end of the tracrRNA. A crRNA and a tracrRNA may bejoined into a single unimolecular or chimeric gRNA, for example, bymeans of a four nucleotide (e.g., GAAA) “tetraloop” or “linker” sequencebridging complementary regions of the crRNA (at its 3′ end) and thetracrRNA (at its 5′ end).

As used herein, a “repeat” sequence or region is a nucleotide sequenceat or near the 3′ end of the crRNA which is complementary to ananti-repeat sequence of a tracrRNA.

As used herein, an “anti-repeat” sequence or region is a nucleotidesequence at or near the 5′ end of the tracrRNA which is complementary tothe repeat sequence of a crRNA.

Additional details regarding guide RNA structure and function, includingthe gRNA/Cas9 complex for genome editing may be found in, at least, Maliet al. Science, 339(6121), 823-826 (2013); Jiang et al. Nat. Biotechnol.31(3). 233-239 (2013); and Jinek et al. Science, 337(6096), 816-821(2012); which are incorporated by reference herein.

As used herein, a “guide sequence” or “targeting sequence” refers to thenucleotide sequence of a gRNA, whether unimolecular or modular, that isfully or partially complementary to a target domain or targetpolynucleotide within a DNA sequence in the genome of a cell whereediting is desired. Guide sequences are typically 10-30 nucleotides inlength, preferably 16-24 nucleotides in length (for example, 16, 17, 18,19, 20, 21, 22, 23 or 24 nucleotides in length), and are at or near the5′ terminus of a Cas9 gRNA.

As used herein, a “target domain” or “target polynucleotide sequence” or“target sequence” is the DNA sequence in a genome of a cell that iscomplementary to the guide sequence of the gRNA.

In the context of formation of a CRISPR complex, “target sequence”refers to a sequence to which a guide sequence is designed to have somecomplementarity, where hybridization between a target sequence and aguide sequence promotes the formation of a CRISPR complex. Fullcomplementarity is not necessarily required, provided there issufficient complementarity to cause hybridization and promote formationof a CRISPR complex. A target sequence may comprise any polynucleotide,such as DNA or RNA polynucleotides. In certain embodiments, a targetsequence is located in the nucleus or cytoplasm of a cell. In otherembodiments, the target sequence may be within an organelle of aeukaryotic cell, for example, mitochondrion or nucleus. Typically, inthe context of a CRISPR system, formation of a CRISPR complex(comprising a guide sequence hybridized to a target sequence andcomplexed with one or more Cas proteins) results in cleavage of one orboth strands in or near (e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 20, 50 or more base pairs) the target sequence. As with the targetsequence, it is believed that complete complementarity is not needed,provided this is sufficient to be functional.

In certain embodiments, one or more vectors driving expression of one ormore elements of a CRISPR system are introduced into a host cell, suchthat expression of the elements of the CRISPR system direct formation ofa CRISPR complex at one or more target sites. For example, a Casnuclease, a crRNA, and a tracrRNA could each be operably linked toseparate regulatory elements on separate vectors. Alternatively, two ormore of the elements expressed from the same or different regulatoryelements may be combined in a single vector, with one or more additionalvectors providing any components of the CRISPR system not included inthe first vector. CRISPR system elements that are combined in a singlevector may be arranged in any suitable orientation, such as one elementlocated 5′ with respect to (“upstream” of) or 3′ with respect to(“downstream” of) a second element. The coding sequence of one elementmay be located on the same or opposite strand of the coding sequence ofa second element, and oriented in the same or opposite direction. Incertain embodiments, a single promoter drives expression of a transcriptencoding a CRISPR enzyme and one or more of the guide sequence, tracrmate sequence (optionally operably linked to the guide sequence), and atracr sequence embedded within one or more intron sequences (e.g., eachin a different intron, two or more in at least one intron, or all in asingle intron).

In certain embodiments, the CRISPR enzyme is part of a fusion proteincomprising one or more heterologous protein domains (e.g. about or morethan about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition tothe CRISPR enzyme). A CRISPR enzyme fusion protein may comprise anyadditional protein sequence, and optionally a linker sequence betweenany two domains. Examples of protein domains that may be fused to aCRISPR enzyme include, without limitation, epitope tags, reporter genesequences, and protein domains having one or more of the followingactivities: methylase activity, demethylase activity, transcriptionactivation activity, transcription repression activity, transcriptionrelease factor activity, histone modification activity, RNA cleavageactivity and nucleic acid binding activity. Additional domains that mayform part of a fusion protein comprising a CRISPR enzyme are describedin U.S. Patent Appl. Publ. No. US20110059502, incorporated herein byreference. In certain embodiments, a tagged CRISPR enzyme is used toidentify the location of a target sequence.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids in mammalian and non-mammalian cells ortarget tissues. Such methods can be used to administer nucleic acidsencoding components of a CRISPR system to cells in culture, or in a hostorganism. Non-viral vector delivery systems include DNA plasmids, RNA(e.g., a transcript of a vector described herein), naked nucleic acid,and nucleic acid complexed with a delivery vehicle, such as a liposome.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell(Anderson, 1992, Science 256:808-813; and Yu, et al., 1994, Gene Therapy1:13-26).

In some embodiments, the CRISPR/Cas is derived from a type II CRISPR/Cassystem. In other embodiments, the CRISPR/Cas system is derived from aCas9 nuclease. Exemplary Cas9 nucleases that may be used in the presentinvention include, but are not limited to, S. pyogenes Cas9 (SpCas9), S.aureus Cas9 (SaCas9), S. thermophilus Cas9 (StCas9), N. meningitidisCas9 (NmCas9), C. jejuni Cas9 (Cj Cas9), and Geobacillus Cas9 (GeoCas9).

In general, Cas proteins comprise at least one RNA recognition and/orRNA binding domain. RNA recognition and/or RNA binding domains interactwith the guiding RNA. Cas proteins can also comprise nuclease domains(i.e., DNase or RNase domains), DNA binding domains, helicase domains,RNAse domains, protein-protein interaction domains, dimerizationdomains, as well as other domains. The Cas proteins can be modified toincrease nucleic acid binding affinity and/or specificity, alter anenzymatic activity, and/or change another property of the protein. Incertain embodiments, the Cas-like protein of the fusion protein can bederived from a wild type Cas9 protein or fragment thereof. In otherembodiments, the Cas can be derived from modified Cas9 protein. Forexample, the amino acid sequence of the Cas9 protein can be modified toalter one or more properties (e.g., nuclease activity, affinity,stability, and so forth) of the protein. Alternatively, domains of theCas9 protein not involved in RNA-guided cleavage can be eliminated fromthe protein such that the modified Cas9 protein is smaller than the wildtype Cas9 protein. In general, a Cas9 protein comprises at least twonuclease (i.e., DNase) domains. For example, a Cas9 protein can comprisea RuvC-like nuclease domain and a HNH-like nuclease domain. The RuvC andHNH domains work together to cut single strands to make adouble-stranded break in DNA. (Jinek, et al., 2012, Science,337:816-821). In certain embodiments, the Cas9-derived protein can bemodified to contain only one functional nuclease domain (either aRuvC-like or a HNH-like nuclease domain). For example, the Cas9-derivedprotein can be modified such that one of the nuclease domains is deletedor mutated such that it is no longer functional (i.e., the nucleaseactivity is absent). In some embodiments in which one of the nucleasedomains is inactive, the Cas9-derived protein is able to introduce anick into a double-stranded nucleic acid (such protein is termed a“nickase”), but not cleave the double-stranded DNA. In any of theabove-described embodiments, any or all of the nuclease domains can beinactivated by one or more deletion mutations, insertion mutations,and/or substitution mutations using well-known methods, such assite-directed mutagenesis, PCR-mediated mutagenesis, and total genesynthesis, as well as other methods known in the art.

In one non-limiting embodiment, a vector drives the expression of theCRISPR system. The art is replete with suitable vectors that are usefulin the present invention. The vectors to be used are suitable forreplication and, optionally, integration in eukaryotic cells. Typicalvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of thedesired nucleic acid sequence. The vectors of the present invention mayalso be used for nucleic acid standard gene delivery protocols. Methodsfor gene delivery are known in the art (U.S. Pat. Nos. 5,399,346,5,580,859 & 5,589,466, incorporated by reference herein in theirentireties).

Further, the vector may be provided to a cell in the form of a viralvector. Viral vector technology is well known in the art and isdescribed, for example, in Sambrook et al. (4th Edition, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York,2012), and in other virology and molecular biology manuals. Viruses,which are useful as vectors include, but are not limited to,retroviruses, adenoviruses, adeno-associated viruses, herpes viruses,Sindbis virus, gammaretrovirus and lentiviruses. In general, a suitablevector contains an origin of replication functional in at least oneorganism, a promoter sequence, convenient restriction endonucleasesites, and one or more selectable markers (e.g., WO 01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).

In some embodiments, guide RNA(s) and Cas9 can be delivered to a cell asa ribonucleoprotein (RNP) complex (e.g., a Cas9/RNA-protein complex).RNPs are comprised of purified Cas9 protein complexed with gRNA and arewell known in the art to be efficiently delivered to multiple types ofcells, including but not limited to stem cells and immune cells(Addgene, Cambridge, Mass., Mirus Bio LLC, Madison, Wis.). In someembodiments, the Cas9/RNA-protein complex is delivered into a cell byelectroporation.

In some embodiments, a gene edited modified cell of the presentdisclosure is edited using CRISPR/Cas9 to disrupt one or more endogenousgenes in a modified cell (e.g., a modified T cell). In some embodiments,CRISPR/Cas9 is used to disrupt one or more of endogenous TRAC, TRBC,PDCD1, A2AR, B7-H3 (CD276), B7-H4 (VTCN1), BTLA (CD272), CD96, CTLA-4(CD152), IDO, KIR, LAG3, TIGIT, TIM-3, and/or VISTA loci, therebyresulting in the downregulation of TRAC, TRBC, PD-1, A2AR, B7-H3(CD276), B7-H4 (VTCN1), BTLA (CD272), CD96, CTLA-4 (CD152), IDO, KIR,LAG3, TIGIT, TIM-3, and/or VISTA. In some embodiments, CRISPR/Cas9 isused to disrupt one or more of endogenous TRAC, TRBC, PDCD1, and/orTIM-3.

In some embodiments, CRISPR/Cas9 is used to disrupt one or more ofendogenous APOA2, AZI2, Bach2, C10orf129, C1orf141, C1orf64, CCL7,CCNI2, CLCA1, CLNS1A, CLOCK, CYSTM1, DEFA4, FAM124A, FAM86A, GALNT2,GLOD5, GPR35, HRF1, HRG, hsa-mir1273d, hsa-mir-6505, KIF27, Klf4, MYBPH,NDC, NDUFS4, PAGR1, PARVG, PMVK, PRKACB, PRPF39, PSG5, PTGIR, PVRL3,TMEM249, TMEM48, TTC27, USP27X, WDR85, YY1AP, and/or ZNRF2 loci, therebyresulting in the downregulation of APOA2, AZI2, Bach2, C10orf129,C1orf141, C1orf64, CCL7, CCNI2, CLCA1, CLNS1A, CLOCK, CYSTM1, DEFA4,FAM124A, FAM86A, GALNT2, GLOD5, GPR35, HRF1, HRG, hsa-mir1273d,hsa-mir-6505, KIF27, Klf4, MYBPH, NDC, NDUFS4, PAGR1, PARVG, PMVK,PRKACB, PRPF39, PSG5, PTGIR, PVRL3, TMEM249, TMEM48, TTC27, USP27X,WDR85, YY1AP, and/or ZNRF2. In certain exemplary embodiments,CRISPR/Cas9 is used to disrupt one or more of endogenous PAGR1, SIX2,KLF4, USP27X, CEACAM19 and/or C1orf14 loci, thereby resulting in thedownregulation of PAGR1, SIX2, KLF4, USP27X, CEACAM19 and/or C1orf14.Suitable gRNAs for use in disrupting one or more of endogenous PAGR1,SIX2, KLF4, USP27X, CEACAM19 and/or C1orf141 is set forth in Table 1.

TABLE 1 SEQ gRNA ID name gRNA sequence NO: PAGR1 AATCAGTATTTCCGCTGCCG 31PAGR1 TTGTACCTGGGGTGCGTCTC 32 PAGR1 AGGAGCAGATCCTTCGTACC 33 PAGR1CCGGTAAGGCCGAGGACGAG 34 PAGR1 CCCCTCGTCCTCGGCCTTAC 35 PAGR1ATTGACCGGAGACGCACCCC 36 SIX2 GCGGGAATTTGCGGCGCACG 37 SIX2ACCCCGCGAGAAGCGTGAGC 38 SIX2 GAGTGGTCTGGCGTCCCCGA 39 SIX2AACAGCCACAACCCGCTGAA 40 SIX2 TTGCTCCTGCGTGAAGCCGA 41 SIX2CAAGGCACACTACATCGAGG 42 KLF4 GTGGTGGCGCCCTACAACGG 43 KLF4AGCCCGCGTAATCACAAGTG 44 KLF4 GCGCGGCGGCCCGCCGTTGT 45 KLF4TCTTTCTCCACGTTCGCGTC 46 KLF4 CACCCACACTTGTGATTACG 47 KLF4GAGAAGACACTGCGTCAAGC 48 USP27X CGCGGCGCACGACTGCGACG 49 USP27XGTGAGATGTCGTCGCTGTTT 50 USP27X CTCGATGCCAGTTGTAGTAT 51 USP27XGTCCAGTACGTCCTTAATAC 52 USP27X TCTTAAACCGATCGTAAAGC 53 USP27XACTGCTTGCGGAGGTTTACG 54 CEACAM19 CTCTGAGGCCGTTGTATCCC 55 CEACAM19ATACAACGGCCTCAGAGGGA 56 CEACAM19 GATCCCTGGCCCCTCGGAGC 57 CEACAM19CATGTGCTGGGCGTCACTGA 58 CEACAM19 GGCCGCCAGGATCCCAGCGT 59 CEACAM19ATCCTGGCGGCCACCATCAT 60 C1orf141 TCTTGCTACATCCGCGTCTA 61 C1orf141CTTTGATATTGCCTTAGACG 62 C1orf141 GATTCTGTTGGTCTCTTAGA 63 C1orf141AATAAAGAAAGTGAGTCAAC 64 C1orf141 AAGAGACCAACAGAATCCAA 65 C1orf141ACATTTGTTTGAATAAAGAA 66

It will be understood to those of skill in the art that guide RNAsequences may be recited with a thymidine (T) or a uridine (U)nucleotide.

In some embodiments, the present invention provides a modified immunecell or precursor cell thereof, comprising: an insertion and/or deletionin a gene locus encoding for a transcriptional modulator, wherein theinsertion and/or deletion is capable of downregulating gene expressionof the endogenous transcriptional modulator. In some embodiments, theinsertion and/or deletion in a gene locus is a CRISPR-mediated insertionand/or deletion in the gene locus. In some embodiments, the gene locusis any one of the endogenous target genes described herein. Accordingly,the present invention provides a modified immune cell or precursor cellthereof, comprising: a CRISPR-mediated insertion and/or deletion in agene locus encoding for any one of the genes described herein, whereinthe CRISPR-mediated insertion and/or deletion is capable ofdownregulating gene expression of the gene.

In some embodiments, the present invention provides a modified immunecell or precursor cell thereof, comprising: an insertion and/or deletionin a gene locus encoding for a transcriptional modulator, wherein theinsertion and/or deletion is capable of downregulating gene expressionof the endogenous transcriptional modulator; and an exogenous T cellreceptor (TCR) and/or chimeric antigen receptor (CAR) comprisingaffinity for an antigen on a target cell. In some embodiments, theinsertion and/or deletion in a gene locus is a CRISPR-mediated insertionand/or deletion in the gene locus. In some embodiments, the gene locusis any one of the endogenous target genes described herein. Accordingly,the present invention provides a modified immune cell or precursor cellthereof, comprising: a CRISPR-mediated insertion and/or deletion in agene locus encoding for any one of the genes described herein, whereinthe CRISPR-mediated insertion and/or deletion is capable ofdownregulating gene expression of the gene; and an exogenous T cellreceptor (TCR) and/or chimeric antigen receptor (CAR) comprisingaffinity for an antigen on a target cell.

In some embodiments, the present invention provides a modified immunecell or precursor cell thereof, comprising: a CRISPR-mediated insertionand/or deletion in a gene locus encoding for an endogenous APOA2, AZI2,Bach2, C10orf129, C1orf141, C1orf64, CCL7, CCNI2, CLCA1, CLNS1A, CLOCK,CYSTM1, DEFA4, FAM124A, FAM86A, GALNT2, GLOD5, GPR35, HRF1, HRG,hsa-mir1273d, hsa-mir-6505, KIF27, Klf4, MYBPH, NDC, NDUFS4, PAGR1,PARVG, PMVK, PRKACB, PRPF39, PSGS, PTGIR, PVRL3, TMEM249, TMEM48, TTC27,USP27X, WDR85, YY1AP, and/or ZNRF2 loci, wherein the CRISPR-mediatedinsertion and/or deletion is capable of downregulating gene expressionof APOA2, AZI2, Bach2, C10orf129, C1orf141, C1orf64, CCL7, CCNI2, CLCA1,CLNS1A, CLOCK, CYSTM1, DEFA4, FAM124A, FAM86A, GALNT2, GLODS, GPR35,HRF1, HRG, hsa-mir1273d, hsa-mir-6505, KIF27, Klf4, MYBPH, NDC, NDUFS4,PAGR1, PARVG, PMVK, PRKACB, PRPF39, PSGS, PTGIR, PVRL3, TMEM249, TMEM48,TTC27, USP27X, WDR85, YY1AP, and/or ZNRF2.

In some embodiments, the present invention provides a modified immunecell or precursor cell thereof, comprising: a CRISPR-mediated insertionand/or deletion in a gene locus encoding for an endogenous APOA2, AZI2,Bach2, C10orf129, C1orf141, C1orf64, CCL7, CCNI2, CLCA1, CLNS1A, CLOCK,CYSTM1, DEFA4, FAM124A, FAM86A, GALNT2, GLODS, GPR35, HRF1, HRG,hsa-mir1273d, hsa-mir-6505, KIF27, Klf4, MYBPH, NDC, NDUFS4, PAGR1,PARVG, PMVK, PRKACB, PRPF39, PSGS, PTGIR, PVRL3, TMEM249, TMEM48, TTC27,USP27X, WDR85, YY1AP, and/or ZNRF2 loci, wherein the CRISPR-mediatedinsertion and/or deletion is capable of downregulating gene expressionof APOA2, AZI2, Bach2, C10orf129, C1orf141, C1orf64, CCL7, CCNI2, CLCA1,CLNS1A, CLOCK, CYSTM1, DEFA4, FAM124A, FAM86A, GALNT2, GLODS, GPR35,HRF1, HRG, hsa-mir1273d, hsa-mir-6505, KIF27, Klf4, MYBPH, NDC, NDUFS4,PAGR1, PARVG, PMVK, PRKACB, PRPF39, PSGS, PTGIR, PVRL3, TMEM249, TMEM48,TTC27, USP27X, WDR85, YY1AP, and/or ZNRF2; and an exogenous T cellreceptor (TCR) and/or chimeric antigen receptor (CAR) comprisingaffinity for an antigen on a target cell.

Accordingly, a method of genetically editing a modified cell of thepresent disclosure comprises introducing into the cell one or morenucleic acids capable of downregulating gene expression of one or moreendogenous genes selected from APOA2, AZI2, Bach2, C10orf129, C1orf141,C1orf64, CCL7, CCNI2, CLCA1, CLNS1A, CLOCK, CYSTM1, DEFA4, FAM124A,FAM86A, GALNT2, GLOD5, GPR35, HRF1, HRG, hsa-mir1273d, hsa-mir-6505,KIF27, Klf4, MYBPH, NDC, NDUFS4, PAGR1, PARVG, PMVK, PRKACB, PRPF39,PSG5, PTGIR, PVRL3, TMEM249, TMEM48, TTC27, USP27X, WDR85, YY1AP, and/orZNRF2. In one embodiment, a method of genetically editing a modifiedcell of the present disclosure comprises introducing into the cell oneor more nucleic acids capable of downregulating gene expression of oneor more endogenous genes selected from PAGR1, SIX2, KLF4, USP27X,CEACAM19 and C1orf141. In one embodiment, a method of geneticallyediting a modified cell of the present disclosure comprises introducinginto the cell one or more nucleic acids capable of downregulating geneexpression of one or more endogenous genes selected from PAGR1, Klf4,and SIX2. In one embodiment, a method of genetically editing a modifiedcell of the present invention comprises introducing into the cell one ormore nucleic acids capable of downregulating gene expression ofendogenous PAGR1. In one embodiment, a method of genetically editing amodified cell of the present invention comprises introducing into thecell one or more nucleic acids capable of downregulating gene expressionof endogenous Klf4. In one embodiment, a method of genetically editing amodified cell of the present invention comprises introducing into thecell one or more nucleic acids capable of downregulating gene expressionof endogenous SIX2.

In one embodiment, a method for generating a modified cell of thepresent disclosure comprises 1) introducing into the cell a nucleic acidcomprising a nucleic acid sequence encoding an exogenous TCR and/or CAR;and 2) introducing into the cell one or more nucleic acids capable ofdownregulating gene expression of one or more endogenous genes selectedfrom PAGR1, SIX2, KLF4, USP27X, CEACAM19 and C1orf141. In an exemplaryembodiment, a method for generating a modified cell of the presentdisclosure comprises 1) introducing into the cell a nucleic acidcomprising a nucleic acid sequence encoding an exogenous TCR and/or CAR;and 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of PAGR1, SIX2, KLF4, USP27X, CEACAM19and/or C1orf141 (e.g., SEQ ID NOs:31-66). In an exemplary embodiment, amethod for generating a modified T cell of the present disclosurecomprises 1) introducing into the T cell a nucleic acid comprising anucleic acid sequence encoding an exogenous TCR and/or CAR; 2)introducing into the cell a nucleic acid capable of downregulating geneexpression of PAGR1, SIX2, KLF4, USP27X, CEACAM19 and/or C1orf141,wherein the nucleic acid capable of downregulating gene expressioncomprises a targeting sequence that is any one of SEQ ID NOs:31-66.

In one embodiment, a method for generating a modified cell of thepresent disclosure comprises 1) introducing into the cell a nucleic acidcomprising a nucleic acid sequence encoding an exogenous TCR and/or CAR;and 2) introducing into the cell one or more nucleic acids capable ofdownregulating gene expression of one or more endogenous genes selectedfrom PAGR1, K14, and SIX2.

In an exemplary embodiment, a method for generating a modified T cell ofthe present disclosure comprises 1) introducing into the T cell anucleic acid comprising a nucleic acid sequence encoding an exogenousTCR and/or CAR; 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of PAGR1, Klf4, and/or SIX2 (e.g., SEQ IDNOs: 31-38). In an exemplary embodiment, a method for generating amodified T cell of the present disclosure comprises 1) introducing intothe T cell a nucleic acid comprising a nucleic acid sequence encoding anexogenous TCR and/or CAR; 2) introducing into the cell a nucleic acidcapable of downregulating gene expression of PAGR1, Klf4, and/or SIX2,wherein the nucleic acid capable of downregulating gene expressioncomprises a targeting sequence that is any one of SEQ ID NOs:31-48.

In an exemplary embodiment, a method for generating a modified T cell ofthe present disclosure comprises 1) introducing into the T cell anucleic acid comprising a nucleic acid sequence encoding an exogenousTCR and/or CAR; 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of PAGR1 (e.g., SEQ ID NOs:31-36). In anexemplary embodiment, a method for generating a modified T cell of thepresent disclosure comprises 1) introducing into the T cell a nucleicacid comprising a nucleic acid sequence encoding an exogenous TCR and/orCAR; 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of PAGR1, wherein the nucleic acidcapable of downregulating gene expression comprises a targeting sequencethat is any one of SEQ ID NOs:31-36.

In an exemplary embodiment, a method for generating a modified T cell ofthe present disclosure comprises 1) introducing into the T cell anucleic acid comprising a nucleic acid sequence encoding an exogenousTCR and/or CAR; 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of SIX2 (e.g., SEQ ID NOs:37-42). In anexemplary embodiment, a method for generating a modified T cell of thepresent disclosure comprises 1) introducing into the T cell a nucleicacid comprising a nucleic acid sequence encoding an exogenous TCR and/orCAR; 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of SIX2, wherein the nucleic acid capableof downregulating gene expression comprises a targeting sequence that isany one of SEQ ID NOs:37-42.

In an exemplary embodiment, a method for generating a modified T cell ofthe present disclosure comprises 1) introducing into the T cell anucleic acid comprising a nucleic acid sequence encoding an exogenousTCR and/or CAR; 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of Klf4 (e.g., SEQ ID NOs:43-48). In anexemplary embodiment, a method for generating a modified T cell of thepresent disclosure comprises 1) introducing into the T cell a nucleicacid comprising a nucleic acid sequence encoding an exogenous TCR and/orCAR; 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of Klf4, wherein the nucleic acid capableof downregulating gene expression comprises a targeting sequence that isany one of SEQ ID NOs:43-48.

In an exemplary embodiment, a method for generating a modified T cell ofthe present disclosure comprises 1) introducing into the T cell anucleic acid comprising a nucleic acid sequence encoding an exogenousTCR and/or CAR; 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of USP27X (e.g., SEQ ID NOs:49-54). In anexemplary embodiment, a method for generating a modified T cell of thepresent disclosure comprises 1) introducing into the T cell a nucleicacid comprising a nucleic acid sequence encoding an exogenous TCR and/orCAR; 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of USP27X, wherein the nucleic acidcapable of downregulating gene expression comprises a targeting sequencethat is any one of SEQ ID NOs:49-54.

In an exemplary embodiment, a method for generating a modified T cell ofthe present disclosure comprises 1) introducing into the T cell anucleic acid comprising a nucleic acid sequence encoding an exogenousTCR and/or CAR; 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of CEACAM19 (e.g., SEQ ID NOs:55-60). Inan exemplary embodiment, a method for generating a modified T cell ofthe present disclosure comprises 1) introducing into the T cell anucleic acid comprising a nucleic acid sequence encoding an exogenousTCR and/or CAR; 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of CEACAM19, wherein the nucleic acidcapable of downregulating gene expression comprises a targeting sequencethat is any one of SEQ ID NOs: 55-60.

In an exemplary embodiment, a method for generating a modified T cell ofthe present disclosure comprises 1) introducing into the T cell anucleic acid comprising a nucleic acid sequence encoding an exogenousTCR and/or CAR; 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of C1orf141 (e.g., SEQ ID NOs:61-66). Inan exemplary embodiment, a method for generating a modified T cell ofthe present disclosure comprises 1) introducing into the T cell anucleic acid comprising a nucleic acid sequence encoding an exogenousTCR and/or CAR; 2) introducing into the cell a nucleic acid capable ofdownregulating gene expression of C1orf141, wherein the nucleic acidcapable of downregulating gene expression comprises a targeting sequencethat is any one of SEQ ID NOs:61-66.

Non-limiting types of CRISPR-mediated modifications include asubstitution, an insertion, a deletion, and an insertion/deletion(INDEL). The modification can be located in any part of the target site(e.g., an endogenous gene locus of any one of the targeted genesdescribed herein), including but not limited to an exon, a splice donor,or a splice acceptor.

In some aspects, the provided compositions and methods include those inwhich at least or greater than about 50%, 60%, 65%, 70%, 75%, 80%, 85%,90% or 95% of immune cells in a composition of immune cells contain thedesired genetic modification. For example, about 50%, 60%, 65%, 70%,75%, 80%, 85%, 90% or 95% of immune cells in a composition of cells intowhich an agent (e.g. gRNA/Cas9) for knockout or genetic disruption ofendogenous gene (e.g., PAGR1, KLF4, or SIX2) was introduced contain thegenetic disruption; do not express the targeted endogenous polypeptide,do not contain a contiguous and/or functional copy of the targeted gene.In some embodiments, the methods, compositions and cells according tothe present disclosure include those in which at least or greater thanabout 50%, 60%, 65%, 70%. 75%, 80%, 85%, 90% or 95% of cells in acomposition of cells into which an agent (e.g. gRNA/Cas9) for knockoutor genetic disruption of a targeted gene was introduced do not expressthe targeted polypeptide, such as on the surface of the immune cells. Insome embodiments, at least or greater than about 50%, 60%, 65%, 70%,75%, 80%, 85%, 90% or 95% of cells in a composition of cells into whichan agent (e.g. gRNA/Cas9) for knockout or genetic disruption of thetargeted gene was introduced are knocked out in both alleles, i.e.comprise a biallelic deletion, in such percentage of cells.

In some embodiments, provided are compositions and methods in which theCas9-mediated cleavage efficiency (% indel) in or near the targeted gene(e.g. within or about within 100 base pairs, within or about within 50base pairs, or within or about within 25 base pairs or within or aboutwithin 10 base pairs upstream or downstream of the cut site) is at leastor greater than about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% incells of a composition of cells into which an agent (e.g. gRNA/Cas9) forknockout or genetic disruption of a targeted gene has been introduced.

In some embodiments, the provided cells, compositions and methodsresults in a reduction or disruption of signals delivered via theendogenous in at least or greater than about 50%, 60%, 65%, 70%, 75%,80%, 85%, 90% or 95% of cells in a composition of cells into which anagent (e.g. gRNA/Cas9) for knockout or genetic disruption of a targetedgene was introduced.

In some embodiments, compositions according to the provided disclosurethat comprise cells engineered with a recombinant receptor and comprisethe reduction, deletion, elimination, knockout or disruption inexpression of an endogenous receptor (e.g. genetic disruption of PAGR1,KLF4, or SIX2) retain the functional property or activities of thereceptor compared to the receptor expressed in engineered cells of acorresponding or reference composition comprising the receptor but donot comprise the genetic disruption of a gene or express the polypeptidewhen assessed under the same conditions. In some embodiments, theengineered cells of the provided compositions retain a functionalproperty or activity compared to a corresponding or referencecomposition comprising engineered cells in which such are engineeredwith the recombinant receptor but do not comprise the genetic disruptionor express the targeted polypeptide when assessed under the sameconditions. In some embodiments, the cells retain cytotoxicity,proliferation, survival or cytokine secretion compared to such acorresponding or reference composition.

In some embodiments, the immune cells in the composition retain aphenotype of the immune cell or cells compared to the phenotype of cellsin a corresponding or reference composition when assessed under the sameconditions. In some embodiments, cells in the composition include naivecells, effector memory cells, central memory cells, stem central memorycells, effector memory cells, and long-lived effector memory cells. Insome embodiments, the percentage of T cells, or T cells expressing therecombinant receptor (e.g. TCR and/or CAR), and comprising the geneticdisruption of a targeted gene (e.g., PAGR1, KLF4, or SIX2) exhibit anon-activated, long-lived memory or central memory phenotype that is thesame or substantially the same as a corresponding or referencepopulation or composition of cells engineered with the recombinantreceptor but not containing the genetic disruption. In some embodiments,such property, activity or phenotype can be measured in an in vitroassay, such as by incubation of the cells in the presence of an antigentargeted by the TCR and/or CAR, a cell expressing the antigen and/or anantigen-receptor activating substance. In some embodiments, any of theassessed activities, properties or phenotypes can be assessed at variousdays following electroporation or other introduction of the agent, suchas after or up to 3, 4, 5, 6, 7 days. In some embodiments, suchactivity, property or phenotype is retained by at least 80%, 85%, 90%,95% or 100% of the cells in the composition compared to the activity ofa corresponding composition containing cells engineered with therecombinant receptor but not comprising the genetic disruption of thetargeted gene when assessed under the same conditions.

As used herein, reference to a “corresponding composition” or a“corresponding population of immune cells” (also called a “referencecomposition” or a “reference population of cells”) refers to immunecells (e.g., T cells) obtained, isolated, generated, produced and/orincubated under the same or substantially the same conditions, exceptthat the immune cells or population of immune cells were not introducedwith the agent. In some aspects, except for not containing introductionof the agent, such immune cells are treated identically or substantiallyidentically as immune cells that have been introduced with the agent,such that any one or more conditions that can influence the activity orproperties of the cell, including the upregulation or expression of theinhibitory molecule, is not varied or not substantially varied betweenthe cells other than the introduction of the agent.

Methods and techniques for assessing the expression and/or levels of Tcell markers are known in the art. Antibodies and reagents for detectionof such markers are well known in the art, and readily available. Assaysand methods for detecting such markers include, but are not limited to,flow cytometry, including intracellular flow cytometry, ELISA, ELISPOT,cytometric bead array or other multiplex methods, Western Blot and otherimmunoaffinity-based methods. In some embodiments, antigen receptor(e.g. TCR and/or CAR)-expressing cells can be detected by flow cytometryor other immunoaffinity based method for expression of a marker uniqueto such cells, and then such cells can be co-stained for another T cellsurface marker or markers.

In some embodiments, the cells, compositions and methods provide for thedeletion, knockout, disruption, or reduction in expression of the targetgene in immune cells (e.g. T cells) to be adoptively transferred (suchas cells engineered to express an exogenous TCR and/or CAR). In someembodiments, the methods are performed ex vivo on primary cells, such asprimary immune cells (e.g. T cells) from a subject. In some aspects,methods of producing or generating such genetically engineered T cellsinclude introducing into a population of cells containing immune cells(e.g. T cells) one or more nucleic acid encoding a recombinant receptor(e.g. exogenous TCR and/or CAR) and an agent or agents that is capableof disrupting, a gene that encode the endogenous receptor to betargeted. As used herein, the term “introducing” encompasses a varietyof methods of introducing DNA into a cell, either in vitro or in vivo,such methods including transformation, transduction, transfection (e.g.electroporation), and infection. Vectors are useful for introducing DNAencoding molecules into cells. Possible vectors include plasmid vectorsand viral vectors. Viral vectors include retroviral vectors, lentiviralvectors, or other vectors such as adenoviral vectors or adeno-associatedvectors.

The population of cells containing T cells can be cells that have beenobtained from a subject, such as obtained from a peripheral bloodmononuclear cells (PBMC) sample, an unfractionated T cell sample, alymphocyte sample, a white blood cell sample, an apheresis product, or aleukapheresis product. In some embodiments, T cells can be separated orselected to enrich T cells in the population using positive or negativeselection and enrichment methods. In some embodiments, the populationcontains CD4+, CD8+ or CD4+ and CD8+ T cells. In some embodiments, thestep of introducing the nucleic acid encoding a genetically engineeredantigen receptor and the step of introducing the agent (e.g. Cas9/gRNARNP) can occur simultaneously or sequentially in any order. In someembodiments, subsequent to introduction of the exogenous receptor andone or more gene editing agents (e.g. Cas9/gRNA RNP), the cells arecultured or incubated under conditions to stimulate expansion and/orproliferation of cells.

Thus, provided are cells, compositions and methods that enhance immunecell, such as T cell, function in adoptive cell therapy, including thoseoffering improved efficacy, such as by increasing activity and potencyof administered genetically engineered cells, while maintainingpersistence or exposure to the transferred cells over time. In someembodiments, the genetically engineered cells, exhibit increasedexpansion and/or persistence when administered in vivo to a subject, ascompared to certain available methods. In some embodiments, the providedimmune cells exhibit increased persistence when administered in vivo toa subject. In some embodiments, the persistence of geneticallyengineered immune cells, in the subject upon administration is greateras compared to that which would be achieved by alternative methods, suchas those involving administration of cells genetically engineered bymethods in which T cells were not introduced with an agent that reducesexpression of or disrupts a gene encoding an endogenous receptor. Insome embodiments, the persistence is increased at least or about atleast 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold,9-fold, 10-fold, 20-fold, 30-fold, 50-fold, 60-fold, 70-fold, 80-fold,90-fold, 100-fold or more.

In some embodiments, the degree or extent of persistence of administeredcells can be detected or quantified after administration to a subject.For example, in some aspects, quantitative PCR (qPCR) is used to assessthe quantity of cells expressing the exogenous receptor (e.g., TCRand/or CAR) in the blood or serum or organ or tissue (e.g., diseasesite) of the subject. In some aspects, persistence is quantified ascopies of DNA or plasmid encoding the exogenous receptor per microgramof DNA, or as the number of receptor-expressing cells per microliter ofthe sample, e.g., of blood or serum, or per total number of peripheralblood mononuclear cells (PBMCs) or white blood cells or T cells permicroliter of the sample. In some embodiments, flow cytometric assaysdetecting cells expressing the receptor generally using antibodiesspecific for the receptors also can be performed. Cell-based assays mayalso be used to detect the number or percentage of functional cells,such as cells capable of binding to and/or neutralizing and/or inducingresponses, e.g., cytotoxic responses, against cells of the disease orcondition or expressing the antigen recognized by the receptor. In anyof such embodiments, the extent or level of expression of another markerassociated with the exogenous receptor (e.g. exogenous TCR and/or CAR)can be used to distinguish the administered cells from endogenous cellsin a subject.

F. Methods of Producing Genetically Modified Immune Cells

The present disclosure provides methods for producing or generating amodified immune cell or precursor thereof (e.g., a T cell) of theinvention for tumor immunotherapy, e.g., adoptive immunotherapy. Thecells generally are engineered by introducing one or more geneticallyengineered nucleic acid encoding the exogenous receptors (e.g., a TCRand/or CAR). In some embodiments, the cells also are introduced, eithersimultaneously or sequentially with the nucleic acid encoding theexogenous receptor, with an agent (e.g. Cas9/gRNA RNP) that is capableof disrupting a targeted gene (e.g., a gene encoding for PAGR1).

In some embodiments, the nucleic acid encoding an exogenous TCR and/orCAR is inserted into a target site (e.g., endogenous gene locus of anyone of the targeted genes described herein) using homology directedrepair.

As used herein, “homology-directed repair” or “HDR” is a mechanism torepair double stranded DNA breaks in cells. HDR generally relies on theprocess of homologous recombination, whereby stretches of nucleic acidsequence homology are used to repair the double stranded DNA break.During HDR, a strand of the homologous sequence of a nucleic acid donorinvades, or hybridizes, with a resected portion of the cut DNA. A DNApolymerase, using the resected DNA as a primer, elongates the cut DNA,using the invaded donor sequence as a template. After elongation andbreak repair, the new sequence at the site of the cut possess whateversequence was present in the nucleic acid donor used in the repairprocess. The process of HDR is further described in Jasin et al. (ColdSpring Harb. Perspect. Biol. 2013 November; 5(11): a012740),incorporated herein by reference.

In some embodiments, the nucleic acid donor template (e.g., forinsertion of a nucleic acid sequence encoding a TCR and/or CAR) may beemployed with gene editing complexes (e.g., CRISPR/Cas system) to enablegenome engineering at specific nucleotide positions in a homologoustarget nucleic acid of a host cell (e.g., homologous chromosomes thatare compound heterozygous at a particular allele). In some aspects, thedisclosure provides a method for targeted gene editing, the methodcomprising delivering to a cell (e.g., a cell of a disease subject) atleast one component of a recombinant gene-editing complex together withthe nucleic acid donor template, under conditions such that therecombinant gene editing complex induces a genetic lesion (e.g., nick ordouble stranded break) in a target site in the chromosome, and the donortemplate of the invention mediates a repair mechanism (e.g., HDR),thereby repairing the lesion.

In certain embodiments, the nucleic acid donor template (also referredto herein as an exogenous donor DNA sequence) facilitates insertion of anucleic acid sequence encoding a TCR and/or CAR into a target site(e.g., an endogenous gene locus) via homologous recombination.Accordingly, in certain embodiments, the nucleic acid sequence encodinga TCR and/or CAR is inserted into the target site (e.g., an endogenousgene locus) via homologous recombination using an exogenous donor DNAsequence. In certain embodiments, the exogenous donor DNA sequencecomprises a 5′ homologous arm comprising the nucleotide sequence setforth in SEQ ID NO: 162. In certain embodiments, the exogenous donor DNAsequence comprises the nucleotide sequence set forth in SEQ ID NO: 163.In certain embodiments, the exogenous donor DNA sequence comprises a 3′homologous arm comprising the nucleotide sequence set forth in SEQ IDNO: 164. In certain embodiments, the exogenous donor DNA sequencecomprises the nucleotide sequence set forth in SEQ ID NO: 165.

5′ homologous arm (SEQ ID NO: 162):AAGGCACCCGCTGGGTCATGTGGTTCGGAGACGGACATTGAGGCTCCCACAGGAGATGCAGATGTCTGGAAAGCAGAGGGAGGGGATGGGGTGAGAGTGCCAGAGTTCCCAGGCAACAAACTTACCCTCAATGTTCCGGCACTTCTGCCGCACCTCGTACACCAGCCGCTCTGCAAGGGGAGGAGAGCTGGCGTCAGAGGTGCCACCCTCTCCAGAAGCAGGCCAACTACCTCTTGTGCGCTCATCAATAATCTCCTTGACCTTGGGCTTCTCCGCTGTGCTCTTCCGGGGCTTTTTGGCTGGTGGAGGTGGTGCGTAGGCAGCTGCCTCAGGTTCCACCCACATGTCCGTGTACACTTCTTTGTAGGGATTCTTCTCTTCTGGAGGAGGAAAGCAGGTGCCAAGGTCAGGGTCCCAGAAAGCTGGGTGCCCTCATTTACCTTCTGGTGGCTCCAGGCCCTTAGGGCCAGAAGGCTGGAAGCCCCCCAGGGCCCATTCAATCATGGGCTTGTTCTGCACCTCCACGGCCTTGGCAGTGTCACTCTCATCGCTGTCGTGGCACACCGGGAACAGCTTCCCCGCGCGGCTGCTGGCCACCTGGAGGGTGACACGCCAGGGTTGGGGTTGCTCCTCCGAGCTCCCAGCAGGGACACTCACCTGCAGGACCTCGTAGATGGCTTTGCGGTACATGGGCTGCTTGTTGTACGTGGCCTGGTGGAACGCACTGCAAAACGAGCTCAGCGGCATCAGCTTCTCAACACACACCTGGGGGGACAAGCCAGGCCTTGTTTGCCGCCCAGGCTACTGCCAAACCCCACAACTTACCACTGAGAAThPGK promoter-eGFP-WPRE-BghPolyA (SEQ ID NO: 163):GGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAGGGGGATCATCGAATTACCTCTAGAGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGTCGACATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATG 3′ homologous arm (SEQ ID NO: 164):GACCCAGCGGGTGCCTTCAGCTGCTCGGCTCCGGCCCGTCATCCACCAAGACACAATGCGGCCTGGCCACCAGGAGAAGCCCCGCAGTTTCCCCCACACCAGCTCCCCAATGCCAAAGCCCCGGCCGTCCTGGAGCCCCAAGGAGCAGAAATCATTACACTGGCCACGGCTGGTGAAGAAGCCGCTCACCTCGTACTCTGGCTCGTCATCGCCTGCTTTGGTGGCATTCTTGTCCCCAGCATCGGACCCCACGGGCTCAGGCGTGGTAGCCACAGTGGGGGATGCGGGGTCAGTGGGCTGCTGCACAGCAGGAGGGCTGGCCTCCTCCACCTTCTGAGACTCCCCGGGCCCCTGGTTTTCTTCCACAGCATTCATTCCTGCAATGACCTTGGCTTTCTTCTCAGCCTGGGGAAACAAAAAACAAAAAGTCACCTTGGCTGGGGCCCAGGCCAGAAGGCGCCTCACCTCCCTTTTCCAGCGTGCCAGCCACTCGTCCCGCTTGCGCTTGCTGATGTAGTAGGGGTCCCCCGCCTGGAAGGTGAGCCTCGGCATGGGCCGCTGACGGAGGCTGGACTCCCAGCCCAAGCCACCCCGCAGCCGGCCCCGGGAGCCCTAGGACAGAGAGACAGACATTAGGGCATTCCACAGAGCCCCTGGGGGTGGAACACTTGCCTCCATTTTCATGGATTCGATGTTGGTCTCCTTCTGTTCTTTGCCTGTGGAGAGGGAAGAACAAAGGGACCAGTAAGAGGCTGCCCCTGGTGCTGAGGACTCACCCGCTTCTGCAGGGGCTCCTCGGCCCGTCTCCGAACCACATGACCCAGCGGGTGCCTT complete sequence of 5′homologous arm-hPGK promoter-eGFP-WPRE-BghPolyA-3′ homologous arm(SEQ ID NO: 165): AAGGCACCCGCTGGGTCATGTGGTTCGGAGACGGACATTGAGGCTCCCACAGGAGATGCAGATGTCTGGAAAGCAGAGGGAGGGGATGGGGTGAGAGTGCCAGAGTTCCCAGGCAACAAACTTACCCTCAATGTTCCGGCACTTCTGCCGCACCTCGTACACCAGCCGCTCTGCAAGGGGAGGAGAGCTGGCGTCAGAGGTGCCACCCTCTCCAGAAGCAGGCCAACTACCTCTTGTGCGCTCATCAATAATCTCCTTGACCTTGGGCTTCTCCGCTGTGCTCTTCCGGGGCTTTTTGGCTGGTGGAGGTGGTGCGTAGGCAGCTGCCTCAGGTTCCACCCACATGTCCGTGTACACTTCTTTGTAGGGATTCTTCTCTTCTGGAGGAGGAAAGCAGGTGCCAAGGTCAGGGTCCCAGAAAGCTGGGTGCCCTCATTTACCTTCTGGTGGCTCCAGGCCCTTAGGGCCAGAAGGCTGGAAGCCCCCCAGGGCCCATTCAATCATGGGCTTGTTCTGCACCTCCACGGCCTTGGCAGTGTCACTCTCATCGCTGTCGTGGCACACCGGGAACAGCTTCCCCGCGCGGCTGCTGGCCACCTGGAGGGTGACACGCCAGGGTTGGGGTTGCTCCTCCGAGCTCCCAGCAGGGACACTCACCTGCAGGACCTCGTAGATGGCTTTGCGGTACATGGGCTGCTTGTTGTACGTGGCCTGGTGGAACGCACTGCAAAACGAGCTCAGCGGCATCAGCTTCTCAACACACACCTGGGGGGACAAGCCAGGCCTTGTTTGCCGCCCAGGCTACTGCCAAACCCCACAACTTACCACTGAGAATGCGATCGCGGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGCTCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTCCGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTGCTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAAGCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCGACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCAGGGCGCGCCGAGAGCAGCGGCCGGGAAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGGTGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAATCACCGACCTCTCTCCCCAGGGGGATCATCGAATTACCTCTAGAGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGTCGACATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCCTGCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGACCCAGCGGGTGCCTTCAGCTGCTCGGCTCCGGCCCGTCATCCACCAAGACACAATGCGGCCTGGCCACCAGGAGAAGCCCCGCAGTTTCCCCCACACCAGCTCCCCAATGCCAAAGCCCCGGCCGTCCTGGAGCCCCAAGGAGCAGAAATCATTACACTGGCCACGGCTGGTGAAGAAGCCGCTCACCTCGTACTCTGGCTCGTCATCGCCTGCTTTGGTGGCATTCTTGTCCCCAGCATCGGACCCCACGGGCTCAGGCGTGGTAGCCACAGTGGGGGATGCGGGGTCAGTGGGCTGCTGCACAGCAGGAGGGCTGGCCTCCTCCACCTTCTGAGACTCCCCGGGCCCCTGGTTTTCTTCCACAGCATTCATTCCTGCAATGACCTTGGCTTTCTTCTCAGCCTGGGGAAACAAAAAACAAAAAGTCACCTTGGCTGGGGCCCAGGCCAGAAGGCGCCTCACCTCCCTTTTCCAGCGTGCCAGCCACTCGTCCCGCTTGCGCTTGCTGATGTAGTAGGGGTCCCCCGCCTGGAAGGTGAGCCTCGGCATGGGCCGCTGACGGAGGCTGGACTCCCAGCCCAAGCCACCCCGCAGCCGGCCCCGGGAGCCCTAGGACAGAGAGACAGACATTAGGGCATTCCACAGAGCCCCTGGGGGTGGAACACTTGCCTCCATTTTCATGGATTCGATGTTGGTCTCCTTCTGTTCTTTGCCTGTGGAGAGGGAAGAACAAAGGGACCAGTAAGAGGCTGCCCCTGGTGCTGAGGACTCACCCGCTTCTGCAGGGGCTCCTCGGCCCGTCTCCGAACCACATGACCCAGC GGGTGCCTT

In some embodiments, the donor DNA sequence can comprise transcriptionalcontrol elements such as, without limitation, a MND promoter, a CMBpromoter, a EF-1alpha promoter, a PGK promoter. In some embodiments, thedonor DNA sequence can comprise a reporter molecule such as, withoutlimitation, a fluorescent marker (e.g., GFP), an epidermal growth factorreceptor (EGFR), a nerve growth factor receptor (NGFR), an induciblecaspase. Where the donor DNA sequence comprises both the primaryinsertion element (e.g., a nucleic acid sequence encoding a TCR and/orCAR) and a secondary element (e.g., a reporter molecule), coordinatedexpression may be desired. Various methods of coordinated expression ofone or more genes are known in the art. In some embodiments, the primaryinsertion element (e.g., a nucleic acid sequence encoding a TCR and/orCAR) and the secondary insertion element (e.g., GFP) is separated by alinker. A linker for use in the present disclosure allows for multipleproteins to be encoded by the same nucleic acid sequence (e.g., amulticistronic or bicistronic sequence), which are translated as apolyprotein that is dissociated into separate protein components. Forexample, a linker for use in a donor nucleic acid of the presentdisclosure comprising a nucleic acid sequence encoding a TCR and/or CARand a reporter gene, allows for the TCR and/or CAR and the reporter geneproduct to be translated as a polyprotein that is dissociated intoseparate TCR and/or CAR and reporter gene product components. Variouslinkers that can be used are disclosed elsewhere herein, e.g., IRES, ora 2A peptide.

In some embodiments, the exogenous receptor (e.g., TCR and/or CAR) isintroduced into a cell by an expression vector. Expression vectorscomprising a nucleic acid sequence encoding a TCR and/or CAR of thepresent invention are provided herein. Suitable expression vectorsinclude lentivirus vectors, gamma retrovirus vectors, foamy virusvectors, adeno associated virus (AAV) vectors, adenovirus vectors,engineered hybrid viruses, naked DNA, including but not limited totransposon mediated vectors, such as Sleeping Beauty, Piggybak, andIntegrases such as Phi31. Some other suitable expression vectors includeHerpes simplex virus (HSV) and retrovirus expression vectors.

Adenovirus expression vectors are based on adenoviruses, which have alow capacity for integration into genomic DNA but a high efficiency fortransfecting host cells. Adenovirus expression vectors containadenovirus sequences sufficient to: (a) support packaging of theexpression vector and (b) to ultimately express the TCR and/or CAR inthe host cell. In some embodiments, the adenovirus genome is a 36 kb,linear, double stranded DNA, where a foreign DNA sequence (e.g., anucleic acid encoding an exogenous TCR and/or CAR) may be inserted tosubstitute large pieces of adenoviral DNA in order to make theexpression vector of the present invention (see, e.g., Danthinne andImperiale, Gene Therapy (2000) 7(20): 1707-1714).

Another expression vector is based on an adeno associated virus, whichtakes advantage of the adenovirus coupled systems. This AAV expressionvector has a high frequency of integration into the host genome. It caninfect nondividing cells, thus making it useful for delivery of genesinto mammalian cells, for example, in tissue cultures or in vivo. TheAAV vector has a broad host range for infectivity. Details concerningthe generation and use of AAV vectors are described in U.S. Pat. Nos.5,139,941 and 4,797,368.

Retrovirus expression vectors are capable of integrating into the hostgenome, delivering a large amount of foreign genetic material, infectinga broad spectrum of species and cell types and being packaged in specialcell lines. The retroviral vector is constructed by inserting a nucleicacid (e.g., a nucleic acid encoding an exogenous TCR and/or CAR) intothe viral genome at certain locations to produce a virus that isreplication defective. Though the retroviral vectors are able to infecta broad variety of cell types, integration and stable expression of theTCR and/or CAR requires the division of host cells.

Lentiviral vectors are derived from lentiviruses, which are complexretroviruses that, in addition to the common retroviral genes gag, pol,and env, contain other genes with regulatory or structural function(see, e.g., U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples oflentiviruses include the Human Immunodeficiency Viruses (HIV-1, HIV-2)and the Simian Immunodeficiency Virus (SIV). Lentiviral vectors havebeen generated by multiply attenuating the HIV virulence genes, forexample, the genes env, vif, vpr, vpu and nef are deleted making thevector biologically safe. Lentiviral vectors are capable of infectingnon-dividing cells and can be used for both in vivo and ex vivo genetransfer and expression, e.g., of a nucleic acid encoding a TCR and/orCAR (see, e.g., U.S. Pat. No. 5,994,136).

Expression vectors including a nucleic acid of the present disclosurecan be introduced into a host cell by any means known to persons skilledin the art. The expression vectors may include viral sequences fortransfection, if desired. Alternatively, the expression vectors may beintroduced by fusion, electroporation, biolistics, transfection,lipofection, or the like. The host cell may be grown and expanded inculture before introduction of the expression vectors, followed by theappropriate treatment for introduction and integration of the vectors.The host cells are then expanded and may be screened by virtue of amarker present in the vectors. Various markers that may be used areknown in the art, and may include hprt, neomycin resistance, thymidinekinase, hygromycin resistance, etc. As used herein, the terms “cell,”“cell line,” and “cell culture” may be used interchangeably. In someembodiments, the host cell an immune cell or precursor thereof, e.g., aT cell, an NK cell, or an NKT cell.

The present invention also provides genetically engineered cells whichinclude and stably express a TCR and/or CAR of the present disclosure.In some embodiments, the genetically engineered cells are geneticallyengineered T-lymphocytes (T cells), naive T cells (TN), memory T cells(for example, central memory T cells (TCM), effector memory cells(TEM)), natural killer cells (NK cells), and macrophages capable ofgiving rise to therapeutically relevant progeny. In one embodiment, thegenetically engineered cells are autologous cells.

Modified cells (e.g., comprising a TCR and/or CAR) may be produced bystably transfecting host cells with an expression vector including anucleic acid of the present disclosure. Additional methods forgenerating a modified cell of the present disclosure include, withoutlimitation, chemical transformation methods (e.g., using calciumphosphate, dendrimers, liposomes and/or cationic polymers), non-chemicaltransformation methods (e.g., electroporation, optical transformation,gene electrotransfer and/or hydrodynamic delivery) and/or particle-basedmethods (e.g., impalefection, using a gene gun and/or magnetofection).Transfected cells expressing a TCR and/or CAR of the present disclosuremay be expanded ex vivo.

Physical methods for introducing an expression vector into host cellsinclude calcium phosphate precipitation, lipofection, particlebombardment, microinjection, electroporation, and the like. Methods forproducing cells including vectors and/or exogenous nucleic acids arewell-known in the art. See, e.g., Sambrook et al. (2001), MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.Chemical methods for introducing an expression vector into a host cellinclude colloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes.

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform may be used as the onlysolvent since it is more readily evaporated than methanol. “Liposome” isa generic term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). Compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the nucleic acids in thehost cell, a variety of assays may be performed. Such assays include,for example, molecular biology assays well known to those of skill inthe art, such as Southern and Northern blotting, RT-PCR and PCR;biochemistry assays, such as detecting the presence or absence of aparticular peptide, e.g., by immunological means (ELISAs and Westernblots) or by assays described herein to identify agents falling withinthe scope of the invention.

In one embodiment, the nucleic acids introduced into the host cell areRNA. In another embodiment, the RNA is mRNA that comprises in vitrotranscribed RNA or synthetic RNA. The RNA may be produced by in vitrotranscription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA may be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA.

PCR may be used to generate a template for in vitro transcription ofmRNA which is then introduced into cells. Methods for performing PCR arewell known in the art. Primers for use in PCR are designed to haveregions that are substantially complementary to regions of the DNA to beused as a template for the PCR. “Substantially complementary,” as usedherein, refers to sequences of nucleotides where a majority or all ofthe bases in the primer sequence are complementary. Substantiallycomplementary sequences are able to anneal or hybridize with theintended DNA target under annealing conditions used for PCR. The primerscan be designed to be substantially complementary to any portion of theDNA template. For example, the primers can be designed to amplify theportion of a gene that is normally transcribed in cells (the openreading frame), including 5′ and 3′ UTRs. The primers may also bedesigned to amplify a portion of a gene that encodes a particular domainof interest. In one embodiment, the primers are designed to amplify thecoding region of a human cDNA, including all or portions of the 5′ and3′ UTRs. Primers useful for PCR are generated by synthetic methods thatare well known in the art. “Forward primers” are primers that contain aregion of nucleotides that are substantially complementary tonucleotides on the DNA template that are upstream of the DNA sequencethat is to be amplified. “Upstream” is used herein to refer to alocation 5, to the DNA sequence to be amplified relative to the codingstrand. “Reverse primers” are primers that contain a region ofnucleotides that are substantially complementary to a double-strandedDNA template that are downstream of the DNA sequence that is to beamplified. “Downstream” is used herein to refer to a location 3′ to theDNA sequence to be amplified relative to the coding strand.

Chemical structures that have the ability to promote stability and/ortranslation efficiency of the RNA may also be used. The RNA preferablyhas 5′ and 3′ UTRs. In one embodiment, the 5′ UTR is between zero and3000 nucleotides in length. The length of 5′ and 3′ UTR sequences to beadded to the coding region can be altered by different methods,including, but not limited to, designing primers for PCR that anneal todifferent regions of the UTRs. Using this approach, one of ordinaryskill in the art can modify the 5′ and 3′ UTR lengths required toachieve optimal translation efficiency following transfection of thetranscribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany mRNAs is known in the art. In other embodiments the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3′ or 5′ UTRto impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one embodiment, the promoter is a T7polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In one embodiment, the mRNA has both a cap on the 5′ end and a 3′poly(A) tail which determine ribosome binding, initiation of translationand stability mRNA in the cell. On a circular DNA template, forinstance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (size can be 50-5000 T), or after PCR by any other method,including, but not limited to, DNA ligation or in vitro recombination.Poly(A) tails also provide stability to RNAs and reduce theirdegradation. Generally, the length of a poly(A) tail positivelycorrelates with the stability of the transcribed RNA. In one embodiment,the poly(A) tail is between 100 and 5000 adenosines.

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotidesresults in about a two-fold increase in the translation efficiency ofthe RNA. Additionally, the attachment of different chemical groups tothe 3′ end can increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA.

5′ caps also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

In some embodiments, the RNA is electroporated into the cells, such asin vitro transcribed RNA. Any solutes suitable for cell electroporation,which can contain factors facilitating cellular permeability andviability such as sugars, peptides, lipids, proteins, antioxidants, andsurfactants can be included.

In some embodiments, a nucleic acid encoding a TCR and/or CAR of thepresent disclosure will be RNA, e.g., in vitro synthesized RNA. Methodsfor in vitro synthesis of RNA are known in the art; any known method canbe used to synthesize RNA comprising a sequence encoding a TCR and/orCAR. Methods for introducing RNA into a host cell are known in the art.See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNAcomprising a nucleotide sequence encoding a TCR and/or CAR into a hostcell can be carried out in vitro, ex vivo or in vivo. For example, ahost cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can beelectroporated in vitro or ex vivo with RNA comprising a nucleotidesequence encoding a TCR and/or CAR.

The disclosed methods can be applied to the modulation of T cellactivity in basic research and therapy, in the fields of cancer, stemcells, acute and chronic infections, and autoimmune diseases, includingthe assessment of the ability of the genetically modified T cell to killa target cancer cell.

The methods also provide the ability to control the level of expressionover a wide range by changing, for example, the promoter or the amountof input RNA, making it possible to individually regulate the expressionlevel. Furthermore, the PCR-based technique of mRNA production greatlyfacilitates the design of the mRNAs with different structures andcombination of their domains.

One advantage of RNA transfection methods of the invention is that RNAtransfection is essentially transient and a vector-free. An RNAtransgene can be delivered to a lymphocyte and expressed thereinfollowing a brief in vitro cell activation, as a minimal expressingcassette without the need for any additional viral sequences. Underthese conditions, integration of the transgene into the host cell genomeis unlikely. Cloning of cells is not necessary because of the efficiencyof transfection of the RNA and its ability to uniformly modify theentire lymphocyte population.

Genetic modification of T cells with in vitro-transcribed RNA (IVT-RNA)makes use of two different strategies both of which have beensuccessively tested in various animal models. Cells are transfected within vitro-transcribed RNA by means of lipofection or electroporation. Itis desirable to stabilize IVT-RNA using various modifications in orderto achieve prolonged expression of transferred IVT-RNA.

Some IVT vectors are known in the literature which are utilized in astandardized manner as template for in vitro transcription and whichhave been genetically modified in such a way that stabilized RNAtranscripts are produced. Currently protocols used in the art are basedon a plasmid vector with the following structure: a 5′ RNA polymerasepromoter enabling RNA transcription, followed by a gene of interestwhich is flanked either 3′ and/or 5′ by untranslated regions (UTR), anda 3′ polyadenyl cassette containing 50-70 A nucleotides. Prior to invitro transcription, the circular plasmid is linearized downstream ofthe polyadenyl cassette by type II restriction enzymes (recognitionsequence corresponds to cleavage site). The polyadenyl cassette thuscorresponds to the later poly(A) sequence in the transcript. As a resultof this procedure, some nucleotides remain as part of the enzymecleavage site after linearization and extend or mask the poly(A)sequence at the 3′ end. It is not clear, whether this nonphysiologicaloverhang affects the amount of protein produced intracellularly fromsuch a construct.

In another aspect, the RNA construct is delivered into the cells byelectroporation. See, e.g., the formulations and methodology ofelectroporation of nucleic acid constructs into mammalian cells astaught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841A1, US2004/0059285A1, US 2004/0092907A1. The various parameters includingelectric field strength required for electroporation of any known celltype are generally known in the relevant research literature as well asnumerous patents and applications in the field. See e.g., U.S. Pat. Nos.6,678,556, 7,171,264, and 7,173,116. Apparatus for therapeuticapplication of electroporation are available commercially, e.g., theMedPulser™ DNA Electroporation Therapy System (Inovio/Genetronics, SanDiego, Calif.), and are described in patents such as U.S. Pat. Nos.6,567,694; 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482;electroporation may also be used for transfection of cells in vitro asdescribed e.g. in US20070128708A1. Electroporation may also be utilizedto deliver nucleic acids into cells in vitro. Accordingly,electroporation-mediated administration into cells of nucleic acidsincluding expression constructs utilizing any of the many availabledevices and electroporation systems known to those of skill in the artpresents an exciting new means for delivering an RNA of interest to atarget cell.

In some embodiments, the immune cells (e.g. T cells) can be incubated orcultivated prior to, during and/or subsequent to introducing the nucleicacid molecule encoding the exogenous receptor (e.g., the TCR and/or CAR)and the gene editing agent (e.g. Cas9/gRNA RNP). In some embodiments,the cells (e.g. T cells) can be incubated or cultivated prior to, duringor subsequent to the introduction of the nucleic acid molecule encodingthe exogenous receptor, such as prior to, during or subsequent to thetransduction of the cells with a viral vector (e.g. lentiviral vector)encoding the exogenous receptor. In some embodiments, the cells (e.g. Tcells) can be incubated or cultivated prior to, during or subsequent tothe introduction of the gene editing agent (e.g. Cas9/gRNA RNP), such asprior to, during or subsequent to contacting the cells with the agent orprior to, during or subsequent to delivering the agent into the cells,e.g. via electroporation. In some embodiments, the incubation can beboth in the context of introducing the nucleic acid molecule encodingthe exogenous receptor and introducing the gene editing agent, e.g.Cas9/gRNA RNP. In some embodiments, the method includes activating orstimulating cells with a stimulating or activating agent (e.g.anti-CD3/anti-CD28 antibodies) prior to introducing the nucleic acidmolecule encoding the exogenous receptor and the gene editing agent,e.g. Cas9/gRNA RNP.

In some embodiments, introducing the gene editing agent, e.g. Cas9/gRNARNP, is done after introducing the nucleic acid molecule encoding theexogenous receptor. In some embodiments, prior to the introducing of theagent, the cells are allowed to rest, e.g. by removal of any stimulatingor activating agent. In some embodiments, prior to introducing theagent, the stimulating or activating agent and/or cytokines are notremoved. Those of skill in the art will be able to determine the orderin which each of the one or more nucleic acid sequences are introducedinto the host cell.

G. Sources of Immune Cells

Prior to expansion, a source of immune cells is obtained from a subjectfor ex vivo manipulation. Sources of target cells for ex vivomanipulation may also include, e.g., autologous or heterologous donorblood, cord blood, or bone marrow. For example the source of immunecells may be from the subject to be treated with the modified immunecells of the invention, e.g., the subject's blood, the subject's cordblood, or the subject's bone marrow. Non-limiting examples of subjectsinclude humans, dogs, cats, mice, rats, and transgenic species thereof.Preferably, the subject is a human.

Immune cells can be obtained from a number of sources, including blood,peripheral blood mononuclear cells, bone marrow, lymph node tissue,spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cellsare cells of the immune system, such as cells of the innate or adaptiveimmunity, e.g., myeloid or lymphoid cells, including lymphocytes,typically T cells and/or NK cells. Other exemplary cells include stemcells, such as multipotent and pluripotent stem cells, including inducedpluripotent stem cells (iPSCs). In some aspects, the cells are humancells. With reference to the subject to be treated, the cells may beallogeneic and/or autologous. The cells typically are primary cells,such as those isolated directly from a subject and/or isolated from asubject and frozen.

In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell(e.g., a CD8+ naive T cell, central memory T cell, or effector memory Tcell), a CD4+ T cell, a natural killer T cell (NKT cells), a regulatoryT cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell ahematopoietic stem cell, a natural killer cell (NK cell) or a dendriticcell. In some embodiments, the cells are monocytes or granulocytes,e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mastcells, eosinophils, and/or basophils. In an embodiment, the target cellis an induced pluripotent stem (iPS) cell or a cell derived from an iPScell, e.g., an iPS cell generated from a subject, manipulated to alter(e.g., induce a mutation in) or manipulate the expression of one or moretarget genes, and differentiated into, e.g., a T cell, e.g., a CD8+ Tcell (e.g., a CD8+ naive T cell, central memory T cell, or effectormemory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoidprogenitor cell or a hematopoietic stem cell.

In some embodiments, the cells include one or more subsets of T cells orother cell types, such as whole T cell populations, CD4+ cells, CD8+cells, and subpopulations thereof, such as those defined by function,activation state, maturity, potential for differentiation, expansion,recirculation, localization, and/or persistence capacities,antigen-specificity, type of antigen receptor, presence in a particularorgan or compartment, marker or cytokine secretion profile, and/ordegree of differentiation. Among the sub-types and subpopulations of Tcells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells,effector T cells (TEFF), memory T cells and sub-types thereof, such asstem cell memory T (TSCM), central memory T (TCM), effector memory T(TEM), or terminally differentiated effector memory T cells,tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells,helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT)cells, naturally occurring and adaptive regulatory T (Treg) cells,helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9cells, TH22 cells, follicular helper T cells, alpha/beta T cells, anddelta/gamma T cells. In certain embodiments, any number of T cell linesavailable in the art, may be used.

In some embodiments, the methods include isolating immune cells from thesubject, preparing, processing, culturing, and/or engineering them. Insome embodiments, preparation of the engineered cells includes one ormore culture and/or preparation steps. The cells for engineering asdescribed may be isolated from a sample, such as a biological sample,e.g., one obtained from or derived from a subject. In some embodiments,the subject from which the cell is isolated is one having the disease orcondition or in need of a cell therapy or to which cell therapy will beadministered. The subject in some embodiments is a human in need of aparticular therapeutic intervention, such as the adoptive cell therapyfor which cells are being isolated, processed, and/or engineered.Accordingly, the cells in some embodiments are primary cells, e.g.,primary human cells. The samples include tissue, fluid, and othersamples taken directly from the subject, as well as samples resultingfrom one or more processing steps, such as separation, centrifugation,genetic engineering (e.g. transduction with viral vector), washing,and/or incubation. The biological sample can be a sample obtaineddirectly from a biological source or a sample that is processed.Biological samples include, but are not limited to, body fluids, such asblood, plasma, serum, cerebrospinal fluid, synovial fluid, urine andsweat, tissue and organ samples, including processed samples derivedtherefrom.

In some aspects, the sample from which the cells are derived or isolatedis blood or a blood-derived sample, or is or is derived from anapheresis or leukapheresis product. Exemplary samples include wholeblood, peripheral blood mononuclear cells (PBMCs), leukocytes, bonemarrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node,gut associated lymphoid tissue, mucosa associated lymphoid tissue,spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon,kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries,tonsil, or other organ, and/or cells derived therefrom. Samples include,in the context of cell therapy, e.g., adoptive cell therapy, samplesfrom autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, e.g., T celllines. The cells in some embodiments are obtained from a xenogeneicsource, for example, from mouse, rat, non-human primate, and pig. Insome embodiments, isolation of the cells includes one or morepreparation and/or non-affinity based cell separation steps. In someexamples, cells are washed, centrifuged, and/or incubated in thepresence of one or more reagents, for example, to remove unwantedcomponents, enrich for desired components, lyse or remove cellssensitive to particular reagents. In some examples, cells are separatedbased on one or more property, such as density, adherent properties,size, sensitivity and/or resistance to particular components.

In some examples, cells from the circulating blood of a subject areobtained, e.g., by apheresis or leukapheresis. The samples, in someaspects, contain lymphocytes, including T cells, monocytes,granulocytes, B cells, other nucleated white blood cells, red bloodcells, and/or platelets, and in some aspects contains cells other thanred blood cells and platelets. In some embodiments, the blood cellscollected from the subject are washed, e.g., to remove the plasmafraction and to place the cells in an appropriate buffer or media forsubsequent processing steps. In some embodiments, the cells are washedwith phosphate buffered saline (PBS). In some aspects, a washing step isaccomplished by tangential flow filtration (TFF) according to themanufacturer's instructions. In some embodiments, the cells areresuspended in a variety of biocompatible buffers after washing. Incertain embodiments, components of a blood cell sample are removed andthe cells directly resuspended in culture media. In some embodiments,the methods include density-based cell separation methods, such as thepreparation of white blood cells from peripheral blood by lysing the redblood cells and centrifugation through a Percoll or Ficoll gradient.

In one embodiment, immune are obtained cells from the circulating bloodof an individual are obtained by apheresis or leukapheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. The cells collected by apheresis may bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media, such as phosphate buffered saline (PBS) orwash solution lacks calcium and may lack magnesium or may lack many ifnot all divalent cations, for subsequent processing steps. Afterwashing, the cells may be resuspended in a variety of biocompatiblebuffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, theundesirable components of the apheresis sample may be removed and thecells directly resuspended in culture media.

In some embodiments, the isolation methods include the separation ofdifferent cell types based on the expression or presence in the cell ofone or more specific molecules, such as surface markers, e.g., surfaceproteins, intracellular markers, or nucleic acid. In some embodiments,any known method for separation based on such markers may be used. Insome embodiments, the separation is affinity- or immunoaffinity-basedseparation. For example, the isolation in some aspects includesseparation of cells and cell populations based on the cells' expressionor expression level of one or more markers, typically cell surfacemarkers, for example, by incubation with an antibody or binding partnerthat specifically binds to such markers, followed generally by washingsteps and separation of cells having bound the antibody or bindingpartner, from those cells having not bound to the antibody or bindingpartner.

Such separation steps can be based on positive selection, in which thecells having bound the reagents are retained for further use, and/ornegative selection, in which the cells having not bound to the antibodyor binding partner are retained. In some examples, both fractions areretained for further use. In some aspects, negative selection can beparticularly useful where no antibody is available that specificallyidentifies a cell type in a heterogeneous population, such thatseparation is best carried out based on markers expressed by cells otherthan the desired population. The separation need not result in 100%enrichment or removal of a particular cell population or cellsexpressing a particular marker. For example, positive selection of orenrichment for cells of a particular type, such as those expressing amarker, refers to increasing the number or percentage of such cells, butneed not result in a complete absence of cells not expressing themarker. Likewise, negative selection, removal, or depletion of cells ofa particular type, such as those expressing a marker, refers todecreasing the number or percentage of such cells, but need not resultin a complete removal of all such cells.

In some examples, multiple rounds of separation steps are carried out,where the positively or negatively selected fraction from one step issubjected to another separation step, such as a subsequent positive ornegative selection. In some examples, a single separation step candeplete cells expressing multiple markers simultaneously, such as byincubating cells with a plurality of antibodies or binding partners,each specific for a marker targeted for negative selection. Likewise,multiple cell types can simultaneously be positively selected byincubating cells with a plurality of antibodies or binding partnersexpressed on the various cell types.

In some embodiments, one or more of the T cell populations is enrichedfor or depleted of cells that are positive for (marker+) or express highlevels (marker^(high)) of one or more particular markers, such assurface markers, or that are negative for (marker -) or expressrelatively low levels (marker^(low)) of one or more markers. Forexample, in some aspects, specific subpopulations of T cells, such ascells positive or expressing high levels of one or more surface markers,e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/orCD45RO+ T cells, are isolated by positive or negative selectiontechniques. In some cases, such markers are those that are absent orexpressed at relatively low levels on certain populations of T cells(such as non-memory cells) but are present or expressed at relativelyhigher levels on certain other populations of T cells (such as memorycells). In one embodiment, the cells (such as the CD8+ cells or the Tcells, e.g., CD3+ cells) are enriched for (i.e., positively selectedfor) cells that are positive or expressing high surface levels ofCD45RO, CCR7, CD28, CD27, CD44, CD 127, and/or CD62L and/or depleted of(e.g., negatively selected for) cells that are positive for or expresshigh surface levels of CD45RA. In some embodiments, cells are enrichedfor or depleted of cells positive or expressing high surface levels ofCD 122, CD95, CD25, CD27, and/or IL7-Ra (CD 127). In some examples, CD8+T cells are enriched for cells positive for CD45RO (or negative forCD45RA) and for CD62L. For example, CD3+, CD28+ T cells can bepositively selected using CD3/CD28 conjugated magnetic beads (e.g.,DYNABEADS® M-450 CD3/CD28 T Cell Expander).

In some embodiments, T cells are separated from a PBMC sample bynegative selection of markers expressed on non-T cells, such as B cells,monocytes, or other white blood cells, such as CD 14. In some aspects, aCD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+cytotoxic T cells. Such CD4+ and CD8+ populations can be further sortedinto sub-populations by positive or negative selection for markersexpressed or expressed to a relatively higher degree on one or morenaive, memory, and/or effector T cell subpopulations. In someembodiments, CD8+ cells are further enriched for or depleted of naive,central memory, effector memory, and/or central memory stem cells, suchas by positive or negative selection based on surface antigensassociated with the respective subpopulation. In some embodiments,enrichment for central memory T (TCM) cells is carried out to increaseefficacy, such as to improve long-term survival, expansion, and/orengraftment following administration, which in some aspects isparticularly robust in such sub-populations. In some embodiments,combining TCM-enriched CD8+ T cells and CD4+ T cells further enhancesefficacy.

In some embodiments, memory T cells are present in both CD62L+ andCD62L-subsets of CD8+ peripheral blood lymphocytes. PBMC can be enrichedfor or depleted of CD62L-CD8+ and/or CD62L+CD8+ fractions, such as usinganti-CD8 and anti-CD62L antibodies. In some embodiments, a CD4+ T cellpopulation and a CD8+ T cell sub-population, e.g., a sub-populationenriched for central memory (TCM) cells. In some embodiments, theenrichment for central memory T (TCM) cells is based on positive or highsurface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; insome aspects, it is based on negative selection for cells expressing orhighly expressing CD45RA and/or granzyme B. In some aspects, isolationof a CD8+ population enriched for TCM cells is carried out by depletionof cells expressing CD4, CD 14, CD45RA, and positive selection orenrichment for cells expressing CD62L. In one aspect, enrichment forcentral memory T (TCM) cells is carried out starting with a negativefraction of cells selected based on CD4 expression, which is subjectedto a negative selection based on expression of CD 14 and CD45RA, and apositive selection based on CD62L. Such selections in some aspects arecarried out simultaneously and in other aspects are carried outsequentially, in either order. In some aspects, the same CD4expression-based selection step used in preparing the CD8+ cellpopulation or subpopulation, also is used to generate the CD4+ cellpopulation or sub-population, such that both the positive and negativefractions from the CD4-based separation are retained and used insubsequent steps of the methods, optionally following one or morefurther positive or negative selection steps.

CD4+T helper cells are sorted into naive, central memory, and effectorcells by identifying cell populations that have cell surface antigens.CD4+ lymphocytes can be obtained by standard methods. In someembodiments, naive CD4+T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+T cells. In some embodiments, central memory CD4+ cells are CD62L+ andCD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO.In one example, to enrich for CD4+ cells by negative selection, amonoclonal antibody cocktail typically includes antibodies to CD14,CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody orbinding partner is bound to a solid support or matrix, such as amagnetic bead or paramagnetic bead, to allow for separation of cells forpositive and/or negative selection.

In some embodiments, the cells are incubated and/or cultured prior to orin connection with genetic engineering. The incubation steps can includeculture, cultivation, stimulation, activation, and/or propagation. Insome embodiments, the compositions or cells are incubated in thepresence of stimulating conditions or a stimulatory agent. Suchconditions include those designed to induce proliferation, expansion,activation, and/or survival of cells in the population, to mimic antigenexposure, and/or to prime the cells for genetic engineering, such as forthe introduction of a recombinant antigen receptor. The conditions caninclude one or more of particular media, temperature, oxygen content,carbon dioxide content, time, agents, e.g., nutrients, amino acids,antibiotics, ions, and/or stimulatory factors, such as cytokines,chemokines, antigens, binding partners, fusion proteins, recombinantsoluble receptors, and any other agents designed to activate the cells.In some embodiments, the stimulating conditions or agents include one ormore agent, e.g., ligand, which is capable of activating anintracellular signaling domain of a TCR complex. In some aspects, theagent turns on or initiates TCR/CD3 intracellular signaling cascade in aT cell. Such agents can include antibodies, such as those specific for aTCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28,for example, bound to solid support such as a bead, and/or one or morecytokines. Optionally, the expansion method may further comprise thestep of adding anti-CD3 and/or anti CD28 antibody to the culture medium(e.g., at a concentration of at least about 0.5 ng/ml). In someembodiments, the stimulating agents include IL-2 and/or IL-15, forexample, an IL-2 concentration of at least about 10 units/mL.

In another embodiment, T cells are isolated from peripheral blood bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient. Alternatively, T cells canbe isolated from an umbilical cord. In any event, a specificsubpopulation of T cells can be further isolated by positive or negativeselection techniques.

The cord blood mononuclear cells so isolated can be depleted of cellsexpressing certain antigens, including, but not limited to, CD34, CD8,CD14, CD19, and CD56. Depletion of these cells can be accomplished usingan isolated antibody, a biological sample comprising an antibody, suchas ascites, an antibody bound to a physical support, and a cell boundantibody.

Enrichment of a T cell population by negative selection can beaccomplished using a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. A preferred method iscell sorting and/or selection via negative magnetic immunoadherence orflow cytometry that uses a cocktail of monoclonal antibodies directed tocell surface markers present on the cells negatively selected. Forexample, to enrich for CD4⁺ cells by negative selection, a monoclonalantibody cocktail typically includes antibodies to CD14, CD20, CD11b,CD16, HLA-DR, and CD8.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion.

T cells can also be frozen after the washing step, which does notrequire the monocyte-removal step. While not wishing to be bound bytheory, the freeze and subsequent thaw step provides a more uniformproduct by removing granulocytes and to some extent monocytes in thecell population. After the washing step that removes plasma andplatelets, the cells may be suspended in a freezing solution. While manyfreezing solutions and parameters are known in the art and will beuseful in this context, in a non-limiting example, one method involvesusing PBS containing 20% DMSO and 8% human serum albumin, or othersuitable cell freezing media. The cells are then frozen to −80° C. at arate of 1° C. per minute and stored in the vapor phase of a liquidnitrogen storage tank. Other methods of controlled freezing may be usedas well as uncontrolled freezing immediately at −20° C. or in liquidnitrogen.

In one embodiment, the population of T cells is comprised within cellssuch as peripheral blood mononuclear cells, cord blood cells, a purifiedpopulation of T cells, and a T cell line. In another embodiment,peripheral blood mononuclear cells comprise the population of T cells.In yet another embodiment, purified T cells comprise the population of Tcells.

In certain embodiments, T regulatory cells (Tregs) can be isolated froma sample. The sample can include, but is not limited to, umbilical cordblood or peripheral blood. In certain embodiments, the Tregs areisolated by flow-cytometry sorting. The sample can be enriched for Tregsprior to isolation by any means known in the art. The isolated Tregs canbe cryopreserved, and/or expanded prior to use. Methods for isolatingTregs are described in U.S. Pat. Nos. 7,754,482, 8,722,400, and9,555,105, and U.S. patent application Ser. No. 13/639,927, contents ofwhich are incorporated herein in their entirety.

H. Expansion of Immune Cells

Whether prior to or after modification of cells to express a TCR and/orCAR, the cells can be activated and expanded in number using methods asdescribed, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055;6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575;7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874;6,797,514; 6,867,041; and U.S. Publication No. 20060121005. For example,the T cells of the invention may be expanded by contact with a surfacehaving attached thereto an agent that stimulates a CD3/TCR complexassociated signal and a ligand that stimulates a co-stimulatory moleculeon the surface of the T cells. In particular, T cell populations may bestimulated by contact with an anti-CD3 antibody, or antigen-bindingfragment thereof, or an anti-CD2 antibody immobilized on a surface, orby contact with a protein kinase C activator (e.g., bryostatin) inconjunction with a calcium ionophore. For co-stimulation of an accessorymolecule on the surface of the T cells, a ligand that binds theaccessory molecule is used. For example, T cells can be contacted withan anti-CD3 antibody and an anti-CD28 antibody, under conditionsappropriate for stimulating proliferation of the T cells. Examples of ananti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon,France) and these can be used in the invention, as can other methods andreagents known in the art (see, e.g., ten Berge et al., Transplant Proc.(1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9):1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2):53-63).

Expanding T cells by the methods disclosed herein can be multiplied byabout 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold,4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater, and anyand all whole or partial integers therebetween. In one embodiment, the Tcells expand in the range of about 20 fold to about 50 fold.

Following culturing, the T cells can be incubated in cell medium in aculture apparatus for a period of time or until the cells reachconfluency or high cell density for optimal passage before passing thecells to another culture apparatus. The culturing apparatus can be ofany culture apparatus commonly used for culturing cells in vitro.Preferably, the level of confluence is 70% or greater before passing thecells to another culture apparatus. More preferably, the level ofconfluence is 90% or greater. A period of time can be any time suitablefor the culture of cells in vitro. The T cell medium may be replacedduring the culture of the T cells at any time. Preferably, the T cellmedium is replaced about every 2 to 3 days. The T cells are thenharvested from the culture apparatus whereupon the T cells can be usedimmediately or cryopreserved to be stored for use at a later time. Inone embodiment, the invention includes cryopreserving the expanded Tcells. The cryopreserved T cells are thawed prior to introducing nucleicacids into the T cell.

In another embodiment, the method comprises isolating T cells andexpanding the T cells. In another embodiment, the invention furthercomprises cryopreserving the T cells prior to expansion. In yet anotherembodiment, the cryopreserved T cells are thawed for electroporationwith the RNA encoding the chimeric membrane protein.

Another procedure for ex vivo expansion cells is described in U.S. Pat.No. 5,199,942 (incorporated herein by reference). Expansion, such asdescribed in U.S. Pat. No. 5,199,942 can be an alternative or inaddition to other methods of expansion described herein. Briefly, exvivo culture and expansion of T cells comprises the addition to thecellular growth factors, such as those described in U.S. Pat. No.5,199,942, or other factors, such as flt3-L, IL-1, IL-3 and c-kitligand. In one embodiment, expanding the T cells comprises culturing theT cells with a factor selected from the group consisting of flt3-L,IL-1, IL-3 and c-kit ligand.

The culturing step as described herein (contact with agents as describedherein or after electroporation) can be very short, for example lessthan 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as describedfurther herein (contact with agents as described herein) can be longer,for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition. A primary cell culture is a culture of cells,tissues or organs taken directly from an organism and before the firstsubculture. Cells are expanded in culture when they are placed in agrowth medium under conditions that facilitate cell growth and/ordivision, resulting in a larger population of the cells. When cells areexpanded in culture, the rate of cell proliferation is typicallymeasured by the amount of time required for the cells to double innumber, otherwise known as the doubling time.

Each round of subculturing is referred to as a passage. When cells aresubcultured, they are referred to as having been passaged. A specificpopulation of cells, or a cell line, is sometimes referred to orcharacterized by the number of times it has been passaged. For example,a cultured cell population that has been passaged ten times may bereferred to as a P10 culture. The primary culture, i.e., the firstculture following the isolation of cells from tissue, is designated P0.Following the first subculture, the cells are described as a secondaryculture (P1 or passage 1). After the second subculture, the cells becomea tertiary culture (P2 or passage 2), and so on. It will be understoodby those of skill in the art that there may be many population doublingsduring the period of passaging; therefore the number of populationdoublings of a culture is greater than the passage number. The expansionof cells (i.e., the number of population doublings) during the periodbetween passaging depends on many factors, including but is not limitedto the seeding density, substrate, medium, and time between passaging.

In one embodiment, the cells may be cultured for several hours (about 3hours) to about 14 days or any hourly integer value in between.Conditions appropriate for T cell culture include an appropriate media(e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15,(Lonza)) that may contain factors necessary for proliferation andviability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10,IL-12, IL-15, TGF-beta, and TNF-α or any other additives for the growthof cells known to the skilled artisan. Other additives for the growth ofcells include, but are not limited to, surfactant, plasmanate, andreducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Mediacan include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, andX-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, andvitamins, either serum-free or supplemented with an appropriate amountof serum (or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

The medium used to culture the T cells may include an agent that canco-stimulate the T cells. For example, an agent that can stimulate CD3is an antibody to CD3, and an agent that can stimulate CD28 is anantibody to CD28. A cell isolated by the methods disclosed herein can beexpanded approximately 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold,500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold,3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, orgreater. In one embodiment, the T cells expand in the range of about 20fold to about 50 fold, or more. In one embodiment, human T regulatorycells are expanded via anti-CD3 antibody coated KT64.86 artificialantigen presenting cells (aAPCs). Methods for expanding and activating Tcells can be found in U.S. Pat. Nos. 7,754,482, 8,722,400, and 9,555,105, contents of which are incorporated herein in their entirety.

In one embodiment, the method of expanding the T cells can furthercomprise isolating the expanded T cells for further applications. Inanother embodiment, the method of expanding can further comprise asubsequent electroporation of the expanded T cells followed byculturing. The subsequent electroporation may include introducing anucleic acid encoding an agent, such as a transducing the expanded Tcells, transfecting the expanded T cells, or electroporating theexpanded T cells with a nucleic acid, into the expanded population of Tcells, wherein the agent further stimulates the T cell. The agent maystimulate the T cells, such as by stimulating further expansion,effector function, or another T cell function.

I. Methods of Treatment

The modified cells (e.g., T cells) described herein may be included in acomposition for immunotherapy. The composition may include apharmaceutical composition and further include a pharmaceuticallyacceptable carrier. A therapeutically effective amount of thepharmaceutical composition comprising the modified T cells may beadministered.

In one aspect, the invention includes a method for adoptive celltransfer therapy comprising administering to a subject in need thereof amodified T cell of the present invention. In another aspect, theinvention includes a method of treating a disease or condition in asubject comprising administering to a subject in need thereof apopulation of modified T cells.

Also included is a method of treating a disease or condition in asubject in need thereof comprising administering to the subject agenetically edited modified cell (e.g., genetically edited modified Tcell). In one embodiment, the method of treating a disease or conditionin a subject in need thereof comprises administering to the subject agenetically edited modified cell comprising an exogenous TCR and/or CAR.

Methods for administration of immune cells for adoptive cell therapy areknown and may be used in connection with the provided methods andcompositions. For example, adoptive T cell therapy methods aredescribed, e.g., in US Patent Application Publication No. 2003/0170238to Gruenberg et al; U.S. Pat. No. 4,690,915 to Rosenberg; Rosenberg(2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al.(2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) BiochemBiophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4):e61338. In some embodiments, the cell therapy, e.g., adoptive T celltherapy is carried out by autologous transfer, in which the cells areisolated and/or otherwise prepared from the subject who is to receivethe cell therapy, or from a sample derived from such a subject. Thus, insome aspects, the cells are derived from a subject, e.g., patient, inneed of a treatment and the cells, following isolation and processingare administered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, iscarried out by allogeneic transfer, in which the cells are isolatedand/or otherwise prepared from a subject other than a subject who is toreceive or who ultimately receives the cell therapy, e.g., a firstsubject. In such embodiments, the cells then are administered to adifferent subject, e.g., a second subject, of the same species. In someembodiments, the first and second subjects are genetically identical. Insome embodiments, the first and second subjects are genetically similar.In some embodiments, the second subject expresses the same HLA class orsupertype as the first subject.

In some embodiments, the subject has been treated with a therapeuticagent targeting the disease or condition, e.g. the tumor, prior toadministration of the cells or composition containing the cells. In someaspects, the subject is refractory or non-responsive to the othertherapeutic agent. In some embodiments, the subject has persistent orrelapsed disease, e.g., following treatment with another therapeuticintervention, including chemotherapy, radiation, and/or hematopoieticstem cell transplantation (HSCT), e.g., allogenic HSCT. In someembodiments, the administration effectively treats the subject despitethe subject having become resistant to another therapy.

In some embodiments, the subject is responsive to the other therapeuticagent, and treatment with the therapeutic agent reduces disease burden.In some aspects, the subject is initially responsive to the therapeuticagent, but exhibits a relapse of the disease or condition over time. Insome embodiments, the subject has not relapsed. In some suchembodiments, the subject is determined to be at risk for relapse, suchas at a high risk of relapse, and thus the cells are administeredprophylactically, e.g., to reduce the likelihood of or prevent relapse.In some aspects, the subject has not received prior treatment withanother therapeutic agent.

In some embodiments, the subject has persistent or relapsed disease,e.g., following treatment with another therapeutic intervention,including chemotherapy, radiation, and/or hematopoietic stem celltransplantation (HSCT), e.g., allogenic HSCT. In some embodiments, theadministration effectively treats the subject despite the subject havingbecome resistant to another therapy.

The modified immune cells of the present invention can be administeredto an animal, preferably a mammal, even more preferably a human, totreat a cancer. In addition, the cells of the present invention can beused for the treatment of any condition related to a cancer, especiallya cell-mediated immune response against a tumor cell(s), where it isdesirable to treat or alleviate the disease. The types of cancers to betreated with the modified cells or pharmaceutical compositions of theinvention include, carcinoma, blastoma, and sarcoma, and certainleukemia or lymphoid malignancies, benign and malignant tumors, andmalignancies e.g., sarcomas, carcinomas, and melanomas. Other exemplarycancers include but are not limited breast cancer, prostate cancer,ovarian cancer, cervical cancer, skin cancer, pancreatic cancer,colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma,leukemia, lung cancer, thyroid cancer, and the like. The cancers may benon-solid tumors (such as hematological tumors) or solid tumors. Adulttumors/cancers and pediatric tumors/cancers are also included. In oneembodiment, the cancer is a solid tumor or a hematological tumor. In oneembodiment, the cancer is a carcinoma. In one embodiment, the cancer isa sarcoma. In one embodiment, the cancer is a leukemia. In oneembodiment the cancer is a solid tumor.

Solid tumors are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumors can be benign or malignant.Different types of solid tumors are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumors, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer,testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors(such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma,ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

Carcinomas that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, esophageal carcinoma, hepatocellularcarcinoma, basal cell carcinoma (a form of skin cancer), squamous cellcarcinoma (various tissues), bladder carcinoma, including transitionalcell carcinoma (a malignant neoplasm of the bladder), bronchogeniccarcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma,lung carcinoma, including small cell carcinoma and non-small cellcarcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma,pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostatecarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductalcarcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterinecarcinoma, testicular carcinoma, osteogenic carcinoma, epithelialcarcinoma, and nasopharyngeal carcinoma.

Sarcomas that can be amenable to therapy by a method disclosed hereininclude, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma,leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

In certain exemplary embodiments, the modified immune cells of theinvention are used to treat a myeloma, or a condition related tomyeloma. Examples of myeloma or conditions related thereto include,without limitation, light chain myeloma, non-secretory myeloma,monoclonal gamopathy of undertermined significance (MGUS), plasmacytoma(e.g., solitary, multiple solitary, extramedullary plasmacytoma),amyloidosis, and multiple myeloma. In one embodiment, a method of thepresent disclosure is used to treat multiple myeloma. In one embodiment,a method of the present disclosure is used to treat refractory myeloma.In one embodiment, a method of the present disclosure is used to treatrelapsed myeloma.

In certain exemplary embodiments, the modified immune cells of theinvention are used to treat a melanoma, or a condition related tomelanoma. Examples of melanoma or conditions related thereto include,without limitation, superficial spreading melanoma, nodular melanoma,lentigo maligna melanoma, acral lentiginous melanoma, amelanoticmelanoma, or melanoma of the skin (e.g., cutaneous, eye, vulva, vagina,rectum melanoma). In one embodiment, a method of the present disclosureis used to treat cutaneous melanoma. In one embodiment, a method of thepresent disclosure is used to treat refractory melanoma. In oneembodiment, a method of the present disclosure is used to treat relapsedmelanoma.

In yet other exemplary embodiments, the modified immune cells of theinvention are used to treat a sarcoma, or a condition related tosarcoma. Examples of sarcoma or conditions related thereto include,without limitation, angiosarcoma, chondrosarcoma, Ewing's sarcoma,fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma,liposarcoma, malignant peripheral nerve sheath tumor, osteosarcoma,pleomorphic sarcoma, rhabdomyosarcoma, and synovial sarcoma. In oneembodiment, a method of the present disclosure is used to treat synovialsarcoma. In one embodiment, a method of the present disclosure is usedto treat liposarcoma such as myxoid/round cell liposarcoma,differentiated/dedifferentiated liposarcoma, and pleomorphicliposarcoma. In one embodiment, a method of the present disclosure isused to treat myxoid/round cell liposarcoma. In one embodiment, a methodof the present disclosure is used to treat a refractory sarcoma. In oneembodiment, a method of the present disclosure is used to treat arelapsed sarcoma.

The cells of the invention to be administered may be autologous, withrespect to the subject undergoing therapy.

The administration of the cells of the invention may be carried out inany convenient manner known to those of skill in the art. The cells ofthe present invention may be administered to a subject by aerosolinhalation, injection, ingestion, transfusion, implantation ortransplantation. The compositions described herein may be administeredto a patient transarterially, subcutaneously, intradermally,intratumorally, intranodally, intramedullary, intramuscularly, byintravenous (i.v.) injection, or intraperitoneally. In other instances,the cells of the invention are injected directly into a site ofinflammation in the subject, a local disease site in the subject, alymphnode, an organ, a tumor, and the like.

In some embodiments, the cells are administered at a desired dosage,which in some aspects includes a desired dose or number of cells or celltype(s) and/or a desired ratio of cell types. Thus, the dosage of cellsin some embodiments is based on a total number of cells (or number perkg body weight) and a desired ratio of the individual populations orsub-types, such as the CD4+ to CD8+ ratio. In some embodiments, thedosage of cells is based on a desired total number (or number per kg ofbody weight) of cells in the individual populations or of individualcell types. In some embodiments, the dosage is based on a combination ofsuch features, such as a desired number of total cells, desired ratio,and desired total number of cells in the individual populations.

In some embodiments, the populations or sub-types of cells, such as CD8⁺and CD4⁺ T cells, are administered at or within a tolerated differenceof a desired dose of total cells, such as a desired dose of T cells. Insome aspects, the desired dose is a desired number of cells or a desirednumber of cells per unit of body weight of the subject to whom the cellsare administered, e.g., cells/kg. In some aspects, the desired dose isat or above a minimum number of cells or minimum number of cells perunit of body weight. In some aspects, among the total cells,administered at the desired dose, the individual populations orsub-types are present at or near a desired output ratio (such as CD4⁺ toCD8⁺ ratio), e.g., within a certain tolerated difference or error ofsuch a ratio.

In some embodiments, the cells are administered at or within a tolerateddifference of a desired dose of one or more of the individualpopulations or sub-types of cells, such as a desired dose of CD4+ cellsand/or a desired dose of CD8+ cells. In some aspects, the desired doseis a desired number of cells of the sub-type or population, or a desirednumber of such cells per unit of body weight of the subject to whom thecells are administered, e.g., cells/kg. In some aspects, the desireddose is at or above a minimum number of cells of the population orsubtype, or minimum number of cells of the population or sub-type perunit of body weight. Thus, in some embodiments, the dosage is based on adesired fixed dose of total cells and a desired ratio, and/or based on adesired fixed dose of one or more, e.g., each, of the individualsub-types or sub-populations. Thus, in some embodiments, the dosage isbased on a desired fixed or minimum dose of T cells and a desired ratioof CD4⁺ to CD8⁺ cells, and/or is based on a desired fixed or minimumdose of CD4⁺ and/or CD8⁺ cells.

In certain embodiments, the cells, or individual populations ofsub-types of cells, are administered to the subject at a range of aboutone million to about 100 billion cells, such as, e.g., 1 million toabout 50 billion cells (e.g., about 5 million cells, about 25 millioncells, about 500 million cells, about 1 billion cells, about 5 billioncells, about 20 billion cells, about 30 billion cells, about 40 billioncells, or a range defined by any two of the foregoing values), such asabout 10 million to about 100 billion cells (e.g., about 20 millioncells, about 30 million cells, about 40 million cells, about 60 millioncells, about 70 million cells, about 80 million cells, about 90 millioncells, about 10 billion cells, about 25 billion cells, about 50 billioncells, about 75 billion cells, about 90 billion cells, or a rangedefined by any two of the foregoing values), and in some cases about 100million cells to about 50 billion cells (e.g., about 120 million cells,about 250 million cells, about 350 million cells, about 450 millioncells, about 650 million cells, about 800 million cells, about 900million cells, about 3 billion cells, about 30 billion cells, about 45billion cells) or any value in between these ranges.

In some embodiments, the dose of total cells and/or dose of individualsub-populations of cells is within a range of between at or about 1×10⁵cells/kg to about 1×10¹¹ cells/kg 10⁴ and at or about 10¹¹cells/kilograms (kg) body weight, such as between 10⁵ and 10⁶ cells/kgbody weight, for example, at or about 1×10⁵ cells/kg, 1.5×10⁵ cells/kg,2×10⁵ cells/kg, or 1×10⁶ cells/kg body weight. For example, in someembodiments, the cells are administered at, or within a certain range oferror of, between at or about 10⁴ and at or about 10⁹ T cells/kilograms(kg) body weight, such as between 10⁵ and 10⁶ T cells/kg body weight,for example, at or about 1×10⁵ T cells/kg, 1.5×10⁵ T cells/kg, 2×10⁵ Tcells/kg, or 1×10⁶ T cells/kg body weight. In other exemplaryembodiments, a suitable dosage range of modified cells for use in amethod of the present disclosure includes, without limitation, fromabout 1×10⁵ cells/kg to about 1×10⁶ cells/kg, from about 1×10⁶ cells/kgto about 1×10⁷ cells/kg, from about 1×10⁷ cells/kg about 1×10⁸ cells/kg,from about 1×10⁸ cells/kg about 1×10⁹ cells/kg, from about 1×10⁹cells/kg about 1×10¹⁰ cells/kg, from about 1×10¹⁰ cells/kg about 1×10¹¹cells/kg. In an exemplary embodiment, a suitable dosage for use in amethod of the present disclosure is about 1×10⁸ cells/kg. In anexemplary embodiment, a suitable dosage for use in a method of thepresent disclosure is about 1×10⁷ cells/kg. In other embodiments, asuitable dosage is from about 1×10⁷ total cells to about 5×10⁷ totalcells. In some embodiments, a suitable dosage is from about 1×10⁸ totalcells to about 5×10⁸ total cells. In some embodiments, a suitable dosageis from about 1.4×10⁷ total cells to about 1.1×10⁹ total cells. In anexemplary embodiment, a suitable dosage for use in a method of thepresent disclosure is about 7×10⁹ total cells.

In some embodiments, the cells are administered at or within a certainrange of error of between at or about 10⁴ and at or about 10⁹ CD4⁺and/or CD8⁺ cells/kilograms (kg) body weight, such as between 10⁵ and10⁶ CD4⁺ and/or CD8⁺ cells/kg body weight, for example, at or about1×10⁵ CD4⁺ and/or CD8⁺ cells/kg, 1.5×10⁵ CD4⁺ and/or CD8⁺ cells/kg,2×10⁵ CD4⁺ and/or CD8⁺ cells/kg, or 1×10⁶ CD4⁺ and/or CD8⁺ cells/kg bodyweight. In some embodiments, the cells are administered at or within acertain range of error of, greater than, and/or at least about 1×10⁶,about 2.5×10⁶, about 5×10⁶, about 7.5×10⁶, or about 9×10⁶ CD4⁺ cells,and/or at least about 1×10⁶, about 2.5×10⁶, about 5×10⁶, about 7.5×10⁶,or about 9×10⁶ CD8+ cells, and/or at least about 1×10⁶, about 2.5×10⁶,about 5×10⁶, about 7.5×10⁶, or about 9×10⁶ T cells. In some embodiments,the cells are administered at or within a certain range of error ofbetween about 10⁸ and 10¹² or between about 10¹⁰ and 10″ T cells,between about 10⁸ and 10¹² or between about 10¹⁰ and 10¹¹ CD4⁺ cells,and/or between about 10⁸ and 10¹² or between about 10¹⁰ and 10¹¹ CD8⁺cells.

In some embodiments, the cells are administered at or within a toleratedrange of a desired output ratio of multiple cell populations orsub-types, such as CD4+ and CD8+ cells or sub-types. In some aspects,the desired ratio can be a specific ratio or can be a range of ratios,for example, in some embodiments, the desired ratio (e.g., ratio of CD4⁺to CD8⁺ cells) is between at or about 5:1 and at or about 5:1 (orgreater than about 1:5 and less than about 5:1), or between at or about1:3 and at or about 3:1 (or greater than about 1:3 and less than about3:1), such as between at or about 2:1 and at or about 1:5 (or greaterthan about 1:5 and less than about 2:1, such as at or about 5:1, 4.5:1,4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1,1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6,1:1.7, 1:1.8, 1:1.9:1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In someaspects, the tolerated difference is within about 1%, about 2%, about3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45%, about 50% of the desired ratio,including any value in between these ranges.

In some embodiments, a dose of modified cells is administered to asubject in need thereof, in a single dose or multiple doses. In someembodiments, a dose of modified cells is administered in multiple doses,e.g., once a week or every 7 days, once every 2 weeks or every 14 days,once every 3 weeks or every 21 days, once every 4 weeks or every 28days. In an exemplary embodiment, a single dose of modified cells isadministered to a subject in need thereof. In an exemplary embodiment, asingle dose of modified cells is administered to a subject in needthereof by rapid intravenous infusion.

For the prevention or treatment of disease, the appropriate dosage maydepend on the type of disease to be treated, the type of cells orrecombinant receptors, the severity and course of the disease, whetherthe cells are administered for preventive or therapeutic purposes,previous therapy, the subject's clinical history and response to thecells, and the discretion of the attending physician. The compositionsand cells are in some embodiments suitably administered to the subjectat one time or over a series of treatments.

In some embodiments, the cells are administered as part of a combinationtreatment, such as simultaneously with or sequentially with, in anyorder, another therapeutic intervention, such as an antibody orengineered cell or receptor or agent, such as a cytotoxic or therapeuticagent.

The cells in some embodiments are co-administered with one or moreadditional therapeutic agents or in connection with another therapeuticintervention, either simultaneously or sequentially in any order. Insome contexts, the cells are co-administered with another therapysufficiently close in time such that the cell populations enhance theeffect of one or more additional therapeutic agents, or vice versa. Insome embodiments, the cells are administered prior to the one or moreadditional therapeutic agents. In some embodiments, the cells areadministered after the one or more additional therapeutic agents. Insome embodiments, the one or more additional agents includes a cytokine,such as IL-2, for example, to enhance persistence. In some embodiments,the methods comprise administration of a chemotherapeutic agent.

In certain embodiments, the modified cells of the invention (e.g., amodified cell comprising a TCR and/or CAR) may be administered to asubject in combination with an immune checkpoint antibody (e.g., ananti-PD1, anti-CTLA-4, or anti-PDL1 antibody). For example, the modifiedcell may be administered in combination with an antibody or antibodyfragment targeting, for example, PD-1 (programmed death 1 protein).Examples of anti-PD-1 antibodies include, but are not limited to,pembrolizumab (KEYTRUDA®, formerly lambrolizumab, also known asMK-3475), and nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®) or anantigen-binding fragment thereof. In certain embodiments, the modifiedcell may be administered in combination with an anti-PD-L1 antibody orantigen-binding fragment thereof. Examples of anti-PD-L1 antibodiesinclude, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®,Atezolizumab), and MEDI4736 (Durvalumab, Imfinzi). In certainembodiments, the modified cell may be administered in combination withan anti-CTLA-4 antibody or antigen-binding fragment thereof. An exampleof an anti-CTLA-4 antibody includes, but is not limited to, Ipilimumab(trade name Yervoy). Other types of immune checkpoint modulators mayalso be used including, but not limited to, small molecules, siRNA,miRNA, and CRISPR systems. Immune checkpoint modulators may beadministered before, after, or concurrently with the modified cellcomprising the CAR. In certain embodiments, combination treatmentcomprising an immune checkpoint modulator may increase the therapeuticefficacy of a therapy comprising a modified cell of the presentinvention.

Following administration of the cells, the biological activity of theengineered cell populations in some embodiments is measured, e.g., byany of a number of known methods. Parameters to assess include specificbinding of an engineered or natural T cell or other immune cell toantigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flowcytometry. In certain embodiments, the ability of the engineered cellsto destroy target cells can be measured using any suitable method knownin the art, such as cytotoxicity assays described in, for example,Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Hermanet al. J. Immunological Methods, 285(1): 25-40 (2004). In certainembodiments, the biological activity of the cells is measured byassaying expression and/or secretion of one or more cytokines, such asCD 107a, IFNγ, IL-2, and TNF. In some aspects the biological activity ismeasured by assessing clinical outcome, such as reduction in tumorburden or load.

In certain embodiments, the subject is provided a secondary treatment.

In some embodiments, the subject can be administered conditioningtherapy prior to CAR T cell therapy. In some embodiments, theconditioning therapy comprises administering an effective amount ofcyclophosphamide to the subject. In some embodiments, the conditioningtherapy comprises administering an effective amount of fludarabine tothe subject. In preferred embodiments, the conditioning therapycomprises administering an effective amount of a combination ofcyclophosphamide and fludarabine to the subject. Administration of aconditioning therapy prior to CAR T cell therapy may increase theefficacy of the CAR T cell therapy. Methods of conditioning patients forT cell therapy are described in U.S. Pat. No. 9,855,298, which isincorporated herein by reference in its entirety.

In certain embodiments, the subject is provided a secondary treatment.Secondary treatments include but are not limited to chemotherapy,radiation, surgery, and medications.

In some embodiments, a specific dosage regimen of the present disclosureincludes a lymphodepletion step prior to the administration of themodified T cells. In an exemplary embodiment, the lymphodepletion stepincludes administration of cyclophosphamide and/or fludarabine.

In some embodiments, the lymphodepletion step includes administration ofcyclophosphamide at a dose of between about 200 mg/m²/day and about 2000mg/m²/day (e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day). In anexemplary embodiment, the dose of cyclophosphamide is about 300mg/m²/day. In some embodiments, the lymphodepletion step includesadministration of fludarabine at a dose of between about 20 mg/m²/dayand about 900 mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30 mg/m²/day,or 60 mg/m²/day). In an exemplary embodiment, the dose of fludarabine isabout 30 mg/m²/day.

In some embodiment, the lymphodepletion step includes administration ofcyclophosphamide at a dose of between about 200 mg/m²/day and about 2000mg/m²/day (e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day), andfludarabine at a dose of between about 20 mg/m²/day and about 900mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60mg/m²/day). In an exemplary embodiment, the lymphodepletion stepincludes administration of cyclophosphamide at a dose of about 300mg/m²/day, and fludarabine at a dose of about 30 mg/m²/day.

In an exemplary embodiment, the dosing of cyclophosphamide is 300mg/m²/day over three days, and the dosing of fludarabine is 30 mg/m²/dayover three days.

Dosing of lymphodepletion chemotherapy may be scheduled on Days −6 to −4(with a −1 day window, i.e., dosing on Days −7 to −5) relative to T cell(e.g., CAR-T, TCR-T, a modified T cell, etc.) infusion on Day 0. In anexemplary embodiment, for a subject having cancer, the subject receiveslymphodepleting chemotherapy including 300 mg/m² of cyclophosphamide byintravenous infusion 3 days prior to administration of the modified Tcells. In an exemplary embodiment, for a subject having cancer, thesubject receives lymphodepleting chemotherapy including 300 mg/m² ofcyclophosphamide by intravenous infusion for 3 days prior toadministration of the modified T cells.

In an exemplary embodiment, for a subject having cancer, the subjectreceives lymphodepleting chemotherapy including fludarabine at a dose ofbetween about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20 mg/m²/day,25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day). In an exemplaryembodiment, for a subject having cancer, the subject receiveslymphodepleting chemotherapy including fludarabine at a dose of 30 mg/m²for 3 days.

In an exemplary embodiment, for a subject having cancer, the subjectreceives lymphodepleting chemotherapy including cyclophosphamide at adose of between about 200 mg/m²/day and about 2000 mg/m²/day (e.g., 200mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day), and fludarabine at a doseof between about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day). In an exemplaryembodiment, for a subject having cancer, the subject receiveslymphodepleting chemotherapy including cyclophosphamide at a dose ofabout 300 mg/m²/day, and fludarabine at a dose of 30 mg/m² for 3 days.

Cells of the invention can be administered in dosages and routes and attimes to be determined in appropriate pre-clinical and clinicalexperimentation and trials. Cell compositions may be administeredmultiple times at dosages within these ranges. Administration of thecells of the invention may be combined with other methods useful totreat the desired disease or condition as determined by those of skillin the art.

It is known in the art that one of the adverse effects followinginfusion of CAR T cells is the onset of immune activation, known ascytokine release syndrome (CRS). CRS is immune activation resulting inelevated inflammatory cytokines. CRS is a known on-target toxicity,development of which likely correlates with efficacy. Clinical andlaboratory measures range from mild CRS (constitutional symptoms and/orgrade-2 organ toxicity) to severe CRS (sCRS; grade ≥3 organ toxicity,aggressive clinical intervention, and/or potentially life threatening).Clinical features include: high fever, malaise, fatigue, myalgia,nausea, anorexia, tachycardia/hypotension, capillary leak, cardiacdysfunction, renal impairment, hepatic failure, and disseminatedintravascular coagulation. Dramatic elevations of cytokines includinginterferon-gamma, granulocyte macrophage colony-stimulating factor,IL-10, and IL-6 have been shown following CAR T-cell infusion. One CRSsignature is elevation of cytokines including IL-6 (severe elevation),IFN-gamma, TNF-alpha (moderate), and IL-2 (mild). Elevations inclinically available markers of inflammation including ferritin andC-reactive protein (CRP) have also been observed to correlate with CRS.The presence of CRS generally correlates with expansion and progressiveimmune activation of adoptively transferred cells. It has beendemonstrated that the degree of CRS severity is dictated by diseaseburden at the time of infusion as patients with high tumor burdenexperience a more severe CRS.

Accordingly, the invention provides for, following the diagnosis of CRS,appropriate CRS management strategies to mitigate the physiologicalsymptoms of uncontrolled inflammation without dampening the antitumorefficacy of the engineered cells (e.g., CAR T cells). CRS managementstrategies are known in the art. For example, systemic corticosteroidsmay be administered to rapidly reverse symptoms of sCRS (e.g., grade 3CRS) without compromising initial antitumor response.

In some embodiments, an anti-IL-6R antibody may be administered. Anexample of an anti-IL-6R antibody is the Food and DrugAdministration-approved monoclonal antibody tocilizumab, also known asatlizumab (marketed as Actemra, or RoActemra). Tocilizumab is ahumanized monoclonal antibody against the interleukin-6 receptor(IL-6R). Administration of tocilizumab has demonstrated near-immediatereversal of CRS.

CRS is generally managed based on the severity of the observed syndromeand interventions are tailored as such. CRS management decisions may bebased upon clinical signs and symptoms and response to interventions,not solely on laboratory values alone.

Mild to moderate cases generally are treated with symptom managementwith fluid therapy, non-steroidal anti-inflammatory drug (NSAID) andantihistamines as needed for adequate symptom relief. More severe casesinclude patients with any degree of hemodynamic instability; with anyhemodynamic instability, the administration of tocilizumab isrecommended. The first-line management of CRS may be tocilizumab, insome embodiments, at the labeled dose of 8 mg/kg IV over 60 minutes (notto exceed 800 mg/dose); tocilizumab can be repeated Q8 hours. Ifsuboptimal response to the first dose of tocilizumab, additional dosesof tocilizumab may be considered. Tocilizumab can be administered aloneor in combination with corticosteroid therapy. Patients with continuedor progressive CRS symptoms, inadequate clinical improvement in 12-18hours or poor response to tocilizumab, may be treated with high-dosecorticosteroid therapy, generally hydrocortisone 100 mg IV ormethylprednisolone 1-2 mg/kg. In patients with more severe hemodynamicinstability or more severe respiratory symptoms, patients may beadministered high-dose corticosteroid therapy early in the course of theCRS. CRS management guidance may be based on published standards (Lee etal. (2019) Biol Blood Marrow Transplant,doi.org/10.1016/j.bbmt.2018.12.758; Neelapu et al. (2018) Nat Rev ClinOncology, 15:47; Teachey et al. (2016) Cancer Discov, 6(6):664-679).

Features consistent with Macrophage Activation Syndrome (MAS) orHemophagocytic lymphohistiocytosis (HLH) have been observed in patientstreated with CAR-T therapy (Henter, 2007), coincident with clinicalmanifestations of the CRS. MAS appears to be a reaction to immuneactivation that occurs from the CRS, and should therefore be considereda manifestation of CRS. MAS is similar to HLH (also a reaction to immunestimulation). The clinical syndrome of MAS is characterized by highgrade non-remitting fever, cytopenias affecting at least two of threelineages, and hepatosplenomegaly. It is associated with high serumferritin, soluble interleukin-2 receptor, and triglycerides, and adecrease of circulating natural killer (NK) activity.

The modified immune cells comprising an exogenous TCR and/or CAR of thepresent invention may be used in a method of treatment as describedherein. In some embodiments, the modified immune cells comprise aninsertion and/or deletion in a gene locus that is capable ofdownregulating gene expression of the endogenous gene. In someembodiments, the endogenous gene is a gene that when downregulated,enhances a function of the immune cell comprising an exogenous TCRand/or CAR. For example, without limitation, the endogenous gene is agene that when downregulated, enhances tumor infiltration, tumorkilling, and/or resistance to immunosuppression of the immune cellcomprising an exogenous TCR and/or CAR.

In some embodiments, the insertion and/or deletion in a gene locus iscapable of downregulating the expression of one or more genes selectedfrom the group consisting of C1orf141, CCDC33, CCL7, CEACAM19, KLF4,MFSD5, PAGR1, SIX2, and USP27X. In some embodiments, the insertionand/or deletion in a gene locus is capable of downregulating theexpression of one or more genes selected from the group consisting ofAZI2, C1orf141, CCDC33, CCL7, CEACAM19, KLF4, MFSD5, PAGR1, SIX2, andUSP27X. In some embodiments, the insertion and/or deletion in a genelocus is capable of downregulating the expression of one or more genesselected from the group consisting of KLF4, PAGR1, and SIX2. In someembodiments, the insertion and/or deletion in a gene locus is capable ofdownregulating the expression of PAGR1. In some embodiments, theinsertion and/or deletion in a gene locus is capable of downregulatingthe expression of a PAGR1-associated gene, e.g., ARID1A, ARID3B, ASXL1,DNMT3A, DUSP1, MAP3K8, PAXIP1, PRMT1, SOCS3, or TNFAIP3.

As such, the modified immune cells comprising an exogenous TCR and/orCAR of the present invention when used in a method of treatment asdescribed herein, enhances the ability of the modified immune cells incarrying out their function. Accordingly, the present invention providesa method for enhancing a function of a modified immune cell for use in amethod of treatment as described herein.

J. Pharmaceutical Compositions and Formulations

Also provided are populations of immune cells of the invention,compositions containing such cells and/or enriched for such cells, suchas in which cells expressing the recombinant receptor make up at least50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore of the total cells in the composition or cells of a certain typesuch as T cells or CD8+ or CD4+ cells. Among the compositions arepharmaceutical compositions and formulations for administration, such asfor adoptive cell therapy. Also provided are therapeutic methods foradministering the cells and compositions to subjects, e.g., patients.

Also provided are compositions including the cells for administration,including pharmaceutical compositions and formulations, such as unitdose form compositions including the number of cells for administrationin a given dose or fraction thereof. The pharmaceutical compositions andformulations generally include one or more optional pharmaceuticallyacceptable carrier or excipient. In some embodiments, the compositionincludes at least one additional therapeutic agent.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered. A “pharmaceutically acceptablecarrier” refers to an ingredient in a pharmaceutical formulation, otherthan an active ingredient, which is nontoxic to a subject. Apharmaceutically acceptable carrier includes, but is not limited to, abuffer, excipient, stabilizer, or preservative. In some aspects, thechoice of carrier is determined in part by the particular cell and/or bythe method of administration. Accordingly, there are a variety ofsuitable formulations. For example, the pharmaceutical composition cancontain preservatives. Suitable preservatives may include, for example,methylparaben, propylparaben, sodium benzoate, and benzalkoniumchloride. In some aspects, a mixture of two or more preservatives isused. The preservative or mixtures thereof are typically present in anamount of about 0.0001% to about 2% by weight of the total composition.Carriers are described, e.g., by Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriersare generally nontoxic to recipients at the dosages and concentrationsemployed, and include, but are not limited to: buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG).

Buffering agents in some aspects are included in the compositions.Suitable buffering agents include, for example, citric acid, sodiumcitrate, phosphoric acid, potassium phosphate, and various other acidsand salts. In some aspects, a mixture of two or more buffering agents isused. The buffering agent or mixtures thereof are typically present inan amount of about 0.001% to about 4% by weight of the totalcomposition. Methods for preparing administrable pharmaceuticalcompositions are known. Exemplary methods are described in more detailin, for example, Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

The formulations can include aqueous solutions. The formulation orcomposition may also contain more than one active ingredient useful forthe particular indication, disease, or condition being treated with thecells, preferably those with activities complementary to the cells,where the respective activities do not adversely affect one another.Such active ingredients are suitably present in combination in amountsthat are effective for the purpose intended. Thus, in some embodiments,the pharmaceutical composition further includes other pharmaceuticallyactive agents or drugs, such as chemotherapeutic agents, e.g.,asparaginase, busulfan, carboplatin, cisplatin, daunorubicin,doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate,paclitaxel, rituximab, vinblastine, and/or vincristine. Thepharmaceutical composition in some embodiments contains the cells inamounts effective to treat or prevent the disease or condition, such asa therapeutically effective or prophylactically effective amount.Therapeutic or prophylactic efficacy in some embodiments is monitored byperiodic assessment of treated subjects. The desired dosage can bedelivered by a single bolus administration of the cells, by multiplebolus administrations of the cells, or by continuous infusionadministration of the cells.

Formulations include those for oral, intravenous, intraperitoneal,subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal,sublingual, or suppository administration. In some embodiments, the cellpopulations are administered parenterally. The term “parenteral,” asused herein, includes intravenous, intramuscular, subcutaneous, rectal,vaginal, and intraperitoneal administration. In some embodiments, thecells are administered to the subject using peripheral systemic deliveryby intravenous, intraperitoneal, or subcutaneous injection. Compositionsin some embodiments are provided as sterile liquid preparations, e.g.,isotonic aqueous solutions, suspensions, emulsions, dispersions, orviscous compositions, which may in some aspects be buffered to aselected pH. Liquid preparations are normally easier to prepare thangels, other viscous compositions, and solid compositions. Additionally,liquid compositions are somewhat more convenient to administer,especially by injection. Viscous compositions, on the other hand, can beformulated within the appropriate viscosity range to provide longercontact periods with specific tissues. Liquid or viscous compositionscan comprise carriers, which can be a solvent or dispersing mediumcontaining, for example, water, saline, phosphate buffered saline,polyoi (for example, glycerol, propylene glycol, liquid polyethyleneglycol) and suitable mixtures thereof.

Sterile injectable solutions can be prepared by incorporating the cellsin a solvent, such as in admixture with a suitable carrier, diluent, orexcipient such as sterile water, physiological saline, glucose,dextrose, or the like. The compositions can contain auxiliary substancessuch as wetting, dispersing, or emulsifying agents (e.g.,methylcellulose), pH buffering agents, gelling or viscosity enhancingadditives, preservatives, flavoring agents, and/or colors, dependingupon the route of administration and the preparation desired. Standardtexts may in some aspects be consulted to prepare suitable preparations.

Various additives which enhance the stability and sterility of thecompositions, including antimicrobial preservatives, antioxidants,chelating agents, and buffers, can be added. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, and sorbic acid.Prolonged absorption of the injectable pharmaceutical form can bebrought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

K. Nucleic Acid and Immune Cell Libraries

The present invention provides a nucleic acid library for use in amethod of screening described elsewhere herein. The nucleic acid librarycomprises one or more nucleic acids. In some embodiments, the librarycomprises one or more nucleic acids each comprising a first nucleic acidencoding a unique targeting sequence. The unique targeting sequence maybe any sequence that is capable of targeting any region of an endogenousgene locus. In one embodiment, the unique targeting sequence is capableof targeting an endogenous gene locus and generating an insertion and/ordeletion in the targeted sequence of the endogenous gene locus. In oneembodiment, the nucleic acid library of the present invention comprisesone or more nucleic acids, wherein each of the one or more nucleic acidscomprises a first nucleic acid encoding a unique targeting sequence. Insome embodiments, the nucleic acid library of the present inventioncomprises one or more nucleic acids, wherein each of the one or morenucleic acids comprises a first nucleic acid encoding a unique targetingsequence, and the library comprises at least one nucleic acid encoding aunique targeting sequence that targets each gene (e.g., open readingframe) of a human genome.

There is an estimated 19,000-20,000 genes in the human genome. As such,in some embodiments, a nucleic acid library of the present inventioncomprises at least 19,000-20,000 nucleic acids, each comprising a firstnucleic acid encoding a unique targeting sequence that targets each ofthe 19,000-20,000 genes of the human genome. For example, a nucleic acidlibrary of the present invention comprises over 100 nucleic acids, eachcomprising a first nucleic acid encoding a unique targeting sequencethat targets at least 100 unique genes (e.g., open reading frames) ofthe human genome. For example, a nucleic acid library of the presentinvention comprises over 200, e.g., over 300, 400, 500, 1000, 1500,2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000,13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, nucleicacids, each comprising a first nucleic acid encoding a unique targetingsequence that targets at least 200, e.g., 300, 400, 500, 1000, 1500,2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000,13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000 uniquegenes of the human genome.

Yet in another embodiment, the nucleic acid library of the presentinvention comprises one or more nucleic acids comprising a first nucleicacid encoding a unique targeting sequence that targets one or moreportions of of each of the 19,000-20,000 genes of the human genome.

In such embodiments, the nucleic acid library of the present inventionis said to have one or more times coverage of the human genome. Forexample, each gene of the human genome may be targeted one or moretimes, e.g., 2 or more times, 3 or more times, 4 or more times, 5 ormore times, 10 or more times, 15 or more times, 20 or more times, andthe nucleic acid library of the present invention comprises at least 2times coverage, e.g, at least 3 times coverage, at least 4 timescoverage, at least 5 times coverage, at least 10 times coverage, atleast 15 times coverage, at least 20 times coverage of the human genome.For example, in one embodiment, a nucleic acid library of the presentinvention that comprises at least 3 times coverage of the human genomecomprises over 600, e.g., over 900, 1200, 1500, 3000, 4500, 6000, 9000,12,000, 15,000, 18,000, 21,000, 24,000, 27,000, 30,000, 33,000, 36,000,39,000, 42,000, 45,000, 48,000, 51,000, 54,000, 57,000, 60,000, nucleicacids, each comprising a first nucleic acid encoding a unique targetingsequence that targets at least 200, e.g., 300, 400, 500, 1000, 1500,2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000,13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000 uniquegenes of the human genome, at least three times. In such an embodiment,the targeting sequences that target the same gene target unique regionsof the same gene. In another example, in one embodiment, a nucleic acidlibrary of the present invention that comprises at least 6 timescoverage of the human genome comprises over 1200, e.g., over 1800, 2400,3000, 6000, 9000, 12,000, 18,000, 24,000, 30,000, 36,000, 42,000,48,000, 54,000, 60,000, 66,000, 72,000, 78,000, 84,000, 90,000, 96,000,102,000, 108,000, 114,000, 120,000, nucleic acids, each comprising afirst nucleic acid encoding a unique targeting sequence that targets atleast 200, e.g., 300, 400, 500, 1000, 1500, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000,16,000, 17,000, 18,000, 19,000, 20,000 unique genes of the human genome,at least six times. In this example, at least six unique targetingsequences target at least six unique regions of the same gene (e.g.,open reading frame). The skilled artisan would readily be able todetermine the appropriate level of coverage and the appropriate size ofthe library (e.g., number of unique targets) for use in a screen ofinterest.

The first nucleic acid of a subject nucleic acid library may furthercomprise a promoter sequence in operable linkage with the uniquetargeting sequence. For example, the first nucleic acid of a subjectnucleic acid library may further comprise a U6 promoter sequence inoperable linkage with the unique targeting sequence. The U6 promoterrecruits RNA polymerase III which transcribes, amongst others, smallRNAs, and is useful for driving transcription of a unique targetingsequence. Any promoter sequence that is suitable for drivingtranscription of small RNAs can be operably linked to the uniquetargeting sequence of a first nucleic acid of a subject nucleic acidlibrary. Another example of such a promoter is the H1 promoter.Accordingly, in one embodiment, a nucleic acid library of the presentinvention comprises one or more nucleic acids, each comprising a firstnucleic acid comprising a U6 promoter operably linked to a nucleic acidsequence encoding a unique targeting sequence. A variety of promoterssuitable for driving transcription of unique targeting sequences areknown in the art. The skilled artisan would readily be able to determinewhich promoter to use that suits the needs of the screen of interest.

In some embodiments, a subject nucleic acid library of the presentinvention comprises one or more nucleic acids each comprising a firstnucleic acid encoding for a unique targeting sequence, e.g., a uniqueguide RNA. The unique guide RNA comprises a sequence that issufficiently complementary with a target region of an endogenous gene.In one embodiment, a nucleic acid library of the present invention thatcomprises at least 6 times coverage of the human genome comprises over1200, e.g., over 1800, 2400, 3000, 6000, 9000, 12,000, 18,000, 24,000,30,000, 36,000, 42,000, 48,000, 54,000, 60,000, 66,000, 72,000, 78,000,84,000, 90,000, 96,000, 102,000, 108,000, 114,000, 120,000, nucleicacids, each comprising a first nucleic acid encoding a unique guide RNAthat targets (e.g., is sufficiently complementary to) a unique targetregion of at least 200, e.g., 300, 400, 500, 1000, 1500, 2000, 3000,4000, 5000, 6000, 7000, 8000, 9000, 10,000, 11,000, 12,000, 13,000,14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000 unique genes ofthe human genes, at least six times.

In some embodiments, the library comprises a second nucleic acid thatencodes for an exogenous T cell receptor (TCR) and/or chimeric antigenreceptor (CAR). In some embodiments, the exogenous TCR and/or CARencoded by the second nucleic acid comprises affinity for a targetantigen, e.g., a known target antigen. In some embodiments, theexogenous TCR and/or CAR encoded by the second nucleic acid comprisesaffinity for, e.g., NY-ESO-1 or PSCA.

In some embodiments, the second nucleic acid of a subject nucleic acidlibrary may further comprise an elongation-factor-1-alpha promoter(EF-1α promoter) in operable linkage with the nucleic acid encoding theexogenous TCR and/or CAR. Use of an EF-1α promoter may increase theefficiency in expression of downstream transgenes (e.g., a TCR and/orCAR encoding nucleic acid sequence). Any promoter sequence that issuitable for driving expression of a downstream transgene, e.g., a TCRand/or CAR, can be operably linked to the nucleic acid encoding for theexogenous TCR and/or CAR. In one embodiment, a nucleic acid library ofthe present invention comprises a second nucleic acid comprising anEF-1α promoter operably linked to a nucleic acid sequence encoding foran exogenous TCR and/or CAR. A variety promoters suitable for drivingexpression of downstream transgenes are known in the art. The skilledartisan would readily be able to determine which promoter to use thatsuits the needs of the screen of interest.

In some embodiments, the first and the second nucleic acid of a subjectnucleic acid library each reside on separate nucleic acids. In suchembodiments, a selection marker, e.g., a detectable label or aresistance gene, may be incorporated into the first and/or the secondnucleic acid. In embodiments where the first and the second nucleic acideach reside on separate nucleic acids, when the library is introducedinto a population of cells, the selectable marker may be used todetermine which cells comprise both the first and the second nucleicacid. In some embodiments, the first and the second nucleic acid resideon the same nucleic acid. A selectable marker may be incorporated intosuch a nucleic acid comprising the first and the second nucleic acids.In one embodiment, the selectable marker is a reporter gene, e.g.,comprises a nucleic acid sequence encoding for a reporter protein, e.g.,a fluorescent protein. Such a selectable marker is useful fordetermining which cells have been successfully transformed or transducedwith a nucleic acid of a subject nucleic acid library.

In an exemplary embodiment, the first and the second nucleic acid of asubject nucleic acid library resides on the same nucleic acid. Forexample, see FIG. 1. In one embodiment, a subject nucleic acid librarycomprises one or more nucleic acids, wherein each nucleic acid comprisesa first nucleic acid encoding a unique targeting sequence, and a secondnucleic acid comprising a nucleic acid sequence encoding for anexogenous TCR and/or CAR. In one embodiment, a subject nucleic acidlibrary comprises one or more nucleic acids, wherein each nucleic acidcomprises a first nucleic acid comprising a U6 promoter sequence inoperable linkage to a nucleic acid sequence encoding a unique targetingsequence, and an EF-1α promoter sequence in operable linkage to anucleic acid sequence encoding for an exogenous TCR and/or CAR.

In those embodiments employing viral vectors in a subject nucleic acidlibrary, members of the nucleic acid library are present as viralparticles that house a viral genomic nucleic acid, where the viralgenomic nucleic acid of a given particle member of the library includesboth a vector domain and a subject nucleic acid (e.g., a nucleic acidcomprising a first nucleic acid encoding a unique targeting sequence,and a second nucleic acid comprising a nucleic acid sequence encodingfor an exogenous TCR and/or CAR). Such libraries may be referred to aspackaged viral nucleic acid libraries. Of particular interest in certainembodiments is the use of packaged viral nucleic acid libraries thatemploy viral vector domains that provide for entry of a single member ofa nucleic acid library into a given target cell (e.g., a target immunecell).

Within a packaged viral nucleic acid library of the invention, the viralgenomic nucleic acids of different library members will share commonvector domains. Accordingly, the nucleic acid library members will sharea common vector sequence, such that the sequence of the encapsidatedviral genomic nucleic acids in the library will be substantially, if notcompletely, identical, but for the different members of the subjectnucleic acid library. The sequence of the vector domain may varygreatly, depending on the nature of the vector. In some instances, thevector domain includes sequences necessary for the production ofrecombinant viral constructs in a packaging cell, transduction andreplication of a member of the nucleic acid library in the target cells(e.g., immune cells) and expression of the member of the nucleic acidlibrary (e.g., expression of a guide RNA and a TCR and/or CAR),reporters or other effectors and genes. Generation of the vector domain,as well as subject nucleic acid libraries including the same, can beaccomplished using any suitable genetic engineering techniques,including without limitation, the standard techniques of PCR,oligonucleotide synthesis, restriction endonuclease digestion,site-specific digestion, site-specific recombination, ligation,transformation, plasmid purification, and DNA sequencing.

In some instances, the vector domain is selected from a viral genome ofa virus selected from the group of adenoviral, adeno-associated,vaccinia, herpes, foamy, etc. viruses, where such viruses are commonlyused for gene transfer applications. In some instances, the vectordomain is a retroviral vector region, such that it is a domain derivedfrom a retrovirus. Retroviruses are any virus belonging to the familyRetroviridae, comprising single-stranded RNA animal virusescharacterized by two unique features. First, the genome of a retrovirusis diploid, consisting of two copies of the RNA. Second, this RNA istranscribed by the virion-associated enzyme reverse transcriptase intodouble-stranded DNA. This double-stranded DNA or provirus may then becapable of integrating into the host genome (e.g., the genome of thetarget immune cell). Accordingly, in certain aspects, each member of asubject nucleic acid library is configured to integrate into the genomeof the target immune cell. The integration may be non-specific orspecific to a particular chromosomal location. In certain aspects, theviral vector is designed to integrate at a specific chromosomal siteusing site-specific recombination (e.g., using a Cre-Lox or otherrecombination system), zinc finger endonuclease, CRISPR endonuclease, ata specific site at which the virus from which the vector is derivednaturally integrates, or the like.

In certain aspects, the members of a subject nucleic acid library arenon-integrating vectors, e.g., where each member of a subject nucleicacid library is based on a non-integrating lentiviral, adenoviral oradeno-associated viral vector.

According to certain embodiments, the retroviral vector region is anadeno-associated viral vector region, e.g., a vector derived from anadeno-associated virus (AAV). Any suitable AAV-based vector with anyserotype of interest may be used, including AAV-based vectors described,e.g., in McCarty (2008) Mol. Therapy 16:1648-1656; Nonnenmacher (2012)Gene Therapy 19:649-658; and Jayandharan et al. (2008) Gene Therapy15:1287-1293.

In some embodiments, the retroviral vector region is a lentiviral vectorregion, e.g., a vector derived from a lentivirus. Lentiviruses aremembers of the retrovirus family. Lentivirus vectors may be pseudotypedwith VSV-G, and have been derived from the human immunodeficiency virus(HIV), the etiologic agent of the human acquired immunodeficiencysyndrome (AIDS); visan-maedi, which causes encephalitis (visna) orpneumonia in sheep; the caprine arthritis-encephalitis virus, whichcauses immune deficiency, arthritis, and encephalopathy in goats; equineinfectious anemia virus (EIAV), which causes autoimmune hemolytic anemiaand encephalopathy in horses; feline immunodeficiency virus (Hy), whichcauses immune deficiency in cats; bovine immune deficiency virus (BIV)which causes lymphadenopathy and lymphocytosis in cattle; and simianimmunodeficiency virus (SIV), which causes immune deficiency andencephalopathy in non-human primates. Vectors that are based on HIV mayretain <5% of the parental genome, and <25% of the genome may beincorporated into packaging constructs, which minimizes the possibilityof the generation of revertant replication-competent HIV. The vectorregion may include sequences form the 5′ and 3′ LTRs of a lentivirus. Insome instances, the vector domain includes the R and U5 sequences fromthe 5′ LTR of a lentivirus and an inactivated or self-inactivating 3′LTR from a lentivirus. The LTR sequences may be LTR sequences from anylentivirus from any species. For example, they may be LTR sequences fromHIV, SIV, FIV or BIV. Where desired, the subject viral nucleic acidlibrary may be made up of self-inactivating vectors that containdeletions of the regulatory elements in the downstreamlong-terminal-repeat sequence, eliminating transcription of thepackaging signal that is required for vector mobilization. As such, thevector region may include an inactivated or self-inactivating 3′ LTR.The 3′ LTR may be made self-inactivating by any convenient method. Forexample, the U3 element of the 3′ LTR may contain a deletion of itsenhancer sequence, such as the TATA box, Sp1 and NF-kappa B sites. As aresult of the self-inactivating 3′ LTR, the provirus that is integratedinto the host cell genome will comprise an inactivated 5′ LTR.Optionally, the U3 sequence from the lentiviral 5′ LTR may be replacedwith a promoter sequence in the viral construct. This may increase thetiter of virus recovered from the packaging cell line. An enhancersequence may also be included.

The viral genomic nucleic acids of a subject viral nucleic acid librarymay contain additional elements, where such elements may vary greatly.For example, a reporter gene may be placed in functional relationshipwith the internal promoter, such as the gene for a fluorescent markerprotein. The additional genetic elements can be operably linked with andcontrolled by an independent promoter/enhancer.

In some embodiments, each member of a subject viral nucleic acid librarymay include an effector cassette, e.g., as described in more detailbelow and in co-pending U.S. Provisional Patent Application No.61/644,324 filed on May 8, 2012, the disclosure of which is hereinincorporated by reference. The term “effector” is used to refer to abiochemical molecule that can effect the transcription, translation,expression, processing or function of another molecule or molecules,such as a target gene or the product of a target gene. Effectors may befull-length proteins, protein domains, peptides, single-stranded ordouble-stranded deoxy- or ribo-oligonucleotides, siRNAs, micro RNAs,CRISPR RNAs, ribozymes, antisense RNAs, regulatory RNAs including smallRNAs and non-coding RNAs, or mimetics or analogues thereof. Effectorcassettes of interest include at least an effector sequence, where theeffector sequence may be operationally-linked to a promoter, e.g., forexpression of the effector sequence in a cell that includes the effectorconstruct. Optionally, an effector cassette may include aneffector-specific barcode, e.g., to facilitate identification ofeffector sequence.

The libraries employed in embodiments of the subject methods can beproduced using any convenient protocol. For example, the subject nucleicacid libraries can be generated synthetically or enzymatically by anumber of different protocols, and the appropriate oligonucleotide andpolynucleotide vectors may be purified using standard recombinant DNAtechniques as described in, for example, Sambrook et al., MolecularCloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (2000), and under regulations described in, e.g.,United States Dept. of HHS, National Institute of Health (NIH)Guidelines for Recombinant DNA Research.

In some embodiments, preparing the subject nucleic acid librariesincludes combining a each member of the nucleic acid library with avector construct comprising a vector domain or vector sequence underconditions sufficient to produce transfection plasmids which, upontransfection of a packaging cell, result in the production of viralparticles containing the subject nucleic acid library as part of genomicnucleic acids encapsidated in viral protein shells. To prepare theproduct transfection plasmids used for transfection, each member of asubject nucleic acid library may be inserted into a vector nucleic acid,where any suitable protocol may be employed. Examples of suitableprotocols include, but are not limited to: DNA ligase mediated joining,recombination enzyme mediate joining, using In-Fusion® PCR protocols(Clontech Laboratories, Mountain View, Calif.), Gateway® cloningtechnology (Life Technologies, Carlsbad, Calif.), and the like.

The resultant product transfection plasmids may then be used totransfect a suitable packaging cell line for production of subjectnucleic acid library viral particles. The packaging cell line providesthe viral proteins that are required in trans for the packaging of theviral genomic RNA into viral particles. The packaging cell line may beany cell line that is capable of expressing retroviral proteins,including HEK293, HeLa, D17, MDCK, BHK, NIH3T3, CHO, CrFK, and Cf2Th. Insome embodiments, each member of a subject viral nucleic acid library isused together with a viral reporter construct which may comprise one ormore reporter genes under the control of a constitutive or conditional(regulatable) promoter. The packaging cell line may stably expressnecessary viral proteins. Such a packaging cell line is described, forexample, in U.S. Pat. No. 6,218,181. Alternatively, a packaging cellline may be transiently transfected with plasmids comprising nucleicacids that encode the necessary viral proteins. In another embodiment, apackaging cell line that does not stably express the necessary viralproteins is co-transfected with two or more plasmids. One of theplasmids comprises the viral construct a member of a subject nucleicacid library. The other plasmid(s) comprises nucleic acids encoding theproteins necessary to allow the cells to produce functional virus thatis able to infect the desired host cell. The packaging cell line may notexpress envelope gene products. In this case, the packaging cell linewill package the viral genome into particles that lack an envelopeprotein. As the envelope protein is responsible, in part, for the hostrange of the viral particles, the viruses preferably are pseudotyped. A“pseudotyped” retrovirus is a retroviral particle having an envelopeprotein that is from a virus other than the virus from which the RNAgenome is derived. The envelope protein may be from a differentretrovirus or a non-retrovirus. One envelope protein is the vesicularstomatitis virus G (VSV-G) protein. Thus, the packaging cell line may betransfected with a plasmid that includes sequences encoding amembrane-associated protein, such as VSV-G, that will permit entry ofthe virus into a target cell. One of skill in the art can choose anappropriate pseudo type specific and/or more efficient for the targetcell used. In addition to conferring a specific host range, a chosenpseudotype may permit the virus to be concentrated to a very high titer.Viruses alternatively can be pseudotyped with ecotropic envelopeproteins that limit infection to a specific species.

The present invention also provides a plurality of immune cellscomprising a subject nucleic acid library. In one embodiment, each ofthe plurality of immune cells comprise a member of the subject nucleicacid library, e.g., a single member of the subject nucleic acid librarycomprising a nucleic acid comprising a first nucleic acid encoding for asingle unique targeting sequence, and a second nucleic acid encoding fora TCR and/or CAR. The plurality of immune cells are contacted with thesubject nucleic acid library under transduction conditions.

Transduction of one or more target cells in the plurality of immunecells with a subject viral nucleic acid library may be accomplished byany convenient protocol and may depend, at least in part, on the targetcell type and the viral vectors employed. For example, transduction mayinclude thawing a frozen subject viral nucleic acid library, suspendingthe cellular sample in a cell culture medium (e.g., D-MEM) which may besupplemented with serum (e.g., 10% FBS) and/or a transduction enhancingagent (e.g., hexadimethrine bromide (Polybrene®)), combining the libraryand cell suspension in a cell culture plate, and placing the plate at37° C. in a CO² incubator for a suitable period of time. In certainaspects, the cells are incubated for between 1 and 24 hours, such asbetween 4 and 16 hours, e.g., between 8 and 12 hours.

The transduction conditions may be optimized in order to achievedelivery and expression of a single member of a subject nucleic acidlibrary into a given target cell. For example, in certain aspects,transducing any given target cell with a single member of a subjectviral nucleic acid library is achieved by employing a sufficientlycomplex packaged viral nucleic acid library and carrying out thetransduction step at a suitable multiplicity of infection (MOI), whichis the ratio of infectious agents (e.g., viral particles) to targetcells (e.g., target immune cells). In some embodiments, the transductionis carried out at an MOI of 1 or less, 0.9 or less, 0.8 or less, 0.7 orless, 0.6 or less, 0.5 or less, 0.4 or less, 0.3 or less, 0.2 or less,or 0.1 or less. Of particular interest are transduction conditions thatresult in each of the plurality of immune cells containing one singlemember of a subject viral nucleic acid library. In such an embodiment,each immune cell comprises a nucleic acid comprising a first nucleicacid encoding for a single unique targeting sequence (e.g., a singleunique guide RNA), and second nucleic acid encoding for a TCR and/orCAR.

In some embodiments, each of a plurality of immune cells comprising asubject nucleic acid library is contacted with an editing agent. As usedherein, an “editing agent” refers to any agent that introduces aninsertion and/or deletion in an endogenous gene locus based on a uniquetargeting sequence comprised by the subject nucleic acid library. In oneembodiment, the editing agent is a CRISPR nuclease polypeptide, or anucleic acid that encodes for a CRISPR nuclease. In embodiments wherethe first and the second nucleic acid of a subject nucleic acid libraryreside on the same nucleic acid, the editing agent may be a CRISPRnuclease, or a nucleic acid encoding a CRISPR nuclease. In embodiments,where the first and the second nucleic acid of a subject nucleic acidlibrary reside on separate nucleic acids, the editing agent may be aCRISPR-related system. In such an embodiment, the CRISPR-related systemcomprises the first nucleic acid of a subject nucleic acid library,e.g., the CRISPR-related system comprises a unique targeting sequence.Where each of a plurality of immune cells comprises a member of asubject nucleic acid library and has been contacted with an editingagent, each of the plurality of immune cells comprise an insertionand/or deletion in an endogenous gene locus (as dictated by the uniquetargeting sequence), and an exogenous TCR and/or CAR. Such a pluralityof gene edited, modified immune cells may be referred to herein as agene modified TCR/CAR immune cell (e.g., T cell) library.

In some embodiments, whether the screening method is performed in vitroor in vivo (as described elsewhere herein), the cells are derived fromcell lines, e.g., T cell lines. The cells in some embodiments areobtained from a host organism source, for example, without limitation,from a mouse, a rat, a non-human primate, and a pig.

L. Methods of Screening

The present invention provides a method for identifying a gene (e.g., amethod of screening for a gene) that regulates immune cell function(e.g., a gene that when downregulated, results in an enhanced functionof an immune cell or precursor cell thereof). For example, such a genemay, without limitation, be a gene that normally acts to inhibit afunction of the immune cell, or may be a gene that normally acts toenhance an inhibitor of a function of the immune cell, or may be a genethat normally acts to inhibit an enhancer of a function of the immunecell. The present invention also provides a method for identifying agene that regulates immune cell memory and persistence (e.g., T cellmemory and T cell persistence). In one embodiment, the present inventionprovides a method for identifying a gene that when downregulated,enhances immune cell memory and/or enhances immune cell persistence. Inone embodiment, the present invention provides a method for identifyinga gene that when downregulated, enhances T cell memory and/or enhances Tcell persistence.

In some embodiments, the present invention provides a method foridentifying a gene that regulates immune cell function (e.g., a genethat when downregulated, results in an enhanced function of an immunecell or precursor cell thereof comprising an exogenous T cell receptor(TCR) and/or chimeric antigen receptor (CAR)). Any of the endogenousgenes described elsewhere herein, when downregulated, may result in anenhanced function of an immune cell comprising an exogenous TCR and/orCAR. The identification method comprises introducing into a plurality ofimmune cells (e.g., population of T cells) a subject nucleic acidlibrary as described elsewhere herein. In some embodiments, theplurality of immune cells is a plurality of T cells modified to expressan exogenous TCR and/or CAR having affinity for an antigen. In someembodiments, the plurality of modified immune cells cells are modifiedT-lymphocytes (T cells), naive T cells (TN), memory T cells (forexample, central memory T cells (TCM), effector memory cells (TEM)),natural killer cells (NK cells), and macrophages. In one embodiment, thegenetically engineered cells are autologous cells. In such embodiments,the plurality of modified immune cells is further modified byintroducing a plurality of agents (e.g., gene editing agents) thattarget a plurality of endogenous genes, thereby generating a pluralityof edited immune cells.

In an exemplary embodiment, the plurality of immune cells is a pluralityof T cells modified by a subject nucleic acid library to express anexogenous TCR and/or CAR having affinity for an antigen, and to expressa unique targeting sequence (e.g., unique guide RNA). In such anembodiment, the plurality of modified immune cells is further modifiedby introducing a plurality of gene editing agents (e.g., a CRISPRnuclease) that results in an insertion and/or deletion in the endogenousgene that the unique target sequence targets, thereby generating aplurality of edited immune cells. Each of the edited immune cellscomprises an insertion and/or deletion in a endogenous gene locus (asdictated by the unique targeting sequence) and an exogenous TCR and/orCAR.

A method for identifying a gene that regulates immune cell function(e.g., a gene that when downregulated, results in an enhanced functionof an immune cell or precursor cell thereof), may be performed in vitroor in vivo. In one embodiment, the identification method is performed invitro. An in vitro method for identifying a gene that regulates immunecell function (e.g., a gene that when downregulated, results in anenhanced function of an immune cell or precursor cell thereof (e.g., Tcell)) is schematically illustrated in FIG. 2. As illustrated, an invitro screening method comprises contacting a gene edited, modifiedTCR/CAR immune cell library (e.g., gene edited, modified TCR/CAR T celllibrary) with target tumor cells. In such an embodiment, the TCR/CARthat is comprised by the immune cells comprises affinity for the targettumor cell. For example, the gene edited, modified TCR/CAR T celllibrary comprises T cells that express a TCR/CAR having affinity for aspecific antigen (e.g., a PSCA CAR), and the T cell library is contactedwith target tumor cells expressing the specific antigen (e.g.,PSCA-expressing target tumor cells). The step of contacting the T celllibrary with target tumor cells represents a “challenge,” e.g., a tumorcell challenge. Each challenge results in an enriched TCR/CAR T celllibrary. For example, upon a first challenge, a first enriched TCR/CAR Tcell library may be isolated. Upon one or more challenges, e.g., two ormore challenges, three or more challenges, four or more challenges, fiveor more challenges, a second or more, e.g., a third or more, a fourth ormore, a fifth or more, sixth or more enriched TCR/CAR T cell library maybe isolated. Upon successive challenges, the final enriched TCR/CAR Tcell library may comprise T cells wherein an endogenous gene has beenedited to confer enhanced T cell functions (e.g., T cell persistence, Tcell efficacy).

In one embodiment, the identification method is performed in vivo. An invivo method for identifying a gene that regulates immune cell function(e.g., a gene that when downregulated, results in an enhanced functionof an immune cell or precursor cell thereof (e.g., T cell)) isschematically illustrated in FIG. 3A. As illustrated, an in vivoscreening method comprises infusing a gene edited, modified TCR/CARimmune cell library (e.g., a gene edited, modified TCR/CAR T celllibrary) into a target tumor-bearing model organism. For example, thegene edited, modified TCR/CAR T cell library comprises T cells thatexpress a TCR/CAR having affinity for a specific antigen (e.g., a PSCACAR), and the T cell library is infused into a tumor-bearing organism,wherein the organism has a tumor that expresses the specific antigen(e.g., PSCA expressing tumor). Tumor infiltrating lymphocytes are thenisolated from the infused, tumor-bearing organism, thereby resulting inan enriched TCR/CAR T cell library. The infusion step is akin to the“challenge” step of an in vitro screening method as described herein. Assuch, in some embodiments, multiple infusions (“challenges”) can beperformed successively to further enrich the resulting isolated TCR/CART cell library.

A suitable model organism for use in an in vivo screening method of thepresent invention includes, without limitation, a mouse, a rat, anon-human primate, and a pig. A suitable model organism generallyincludes an organism that has a natural immune cell repertoire (e.g., Tcell repertoire). In some embodiments, where the identification methodis performed in vivo, the gene edited, modified TCR/CAR immune cell(e.g., T cell) library is generated from a population of immune cells(e.g., T cells) that are obtained from the same animal as the subject ofinfusion. For example, where the in vivo screening method includesinfusing a subject gene edited, modified TCR/CAR T cell library into atumor-bearing mouse, the gene edited, modified TCR/CAR T cell librarymay be generated from a population of mouse T cells. In someembodiments, where the identification method is performed in vivo, thegene edited, modified TCR/CAR immune cell (e.g., T cell) library isgenerated from a population of immune cells (e.g., T cells) that areobtained from a different animal as the subject of infusion. Forexample, where the in vivo screening method includes infusing a subjectgene edited, modified TCR/CAR T cell library into a tumor-bearing mouse,the gene edited, modified TCR/CAR T cell library may be generated from apopulation of human T cells. The skilled artisan would readily be ableto determine the appropriate source of immune cells and the appropriateinfusion subject.

An in vivo method for identifying a gene that regulates immune cellmemory and/or immune cell persistence (e.g., a gene that when that whendownregulated, results in enhanced T cell memory and/or T cellpersistence) is schematically illustrated in FIG. 3A. As illustrated, anin vivo screening method comprises infusing a gene edited, modifiedTCR/CAR immune cell library (e.g., a gene edited, modified TCR/CAR Tcell library) into a target tumor-bearing model organism. For example,the gene edited, modified TCR/CAR T cell library comprises T cells thatexpress a TCR/CAR having affinity for a specific antigen (e.g., a PSCACAR), and the T cell library is infused into a tumor-bearing organism,wherein the organism has a tumor that expresses the specific antigen(e.g., PSCA expressing tumor). In one embodiment, the gene edited,modified TCR/CAR T cell library will be able to clear a significantportion of the tumor from the tumor-bearing organism. In one embodiment,the gene edited, modified TCR/CAR T cell library will be able to fullyclear the tumor from the tumor-bearing organism. In one embodiment, thegene edited, modified TCR/CAR T cell library will be able to clear thetumor from the tumor-bearing organism to levels that are undetectableusing standard methods. Once the gene edited, modified TCR/CAR T celllibrary clears the tumor from the tumor-bearing organis, tumorre-inoculation is performed to re-introduce tumor cells into the clearedtumor-bearing organism. The re-inoculated tumor may be controlled bymemory library T cells. Tumor infiltrating lymphocytes are then isolatedfrom the re-inoculated organism, thereby resulting in an enrichedTCR/CAR T cell library.

Once an enriched TCR/CAR immune cell library (e.g., an enriched TCR/CART cell library) is isolated, standard sequencing methods may be used toidentify the gene that regulates immune cell function, immune cellmemory, and/or immune cell persistence (e.g., T cell function, T cellmemory, and/or T cell persistence). Several methods of DNA extractionand analysis are encompassed in the methods of the invention. As usedherein “deep sequencing” indicates that the depth of the process is manytimes larger than the length of the sequence under study. Deepsequencing is encompassed in next generation sequencing methods whichinclude but are not limited to single molecule realtime sequencing(Pacific Bio), Ion semiconductor (Ion torrent sequencing),Pyrosequencing (454), Sequencing by synthesis (Illumina), Sequencing byligations (SOLiD sequencing) and Chain termination (Sanger sequencing).The skilled artisan would be able to determine the targeted gene that isenriched in the enriched TCR/CAR immune cell library.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. It will be readily apparent to those skilled in the art thatother suitable modifications and adaptations of the methods describedherein may be made using suitable equivalents without departing from thescope of the embodiments disclosed herein. In addition, manymodifications may be made to adapt a particular situation, material,composition of matter, process, process step or steps, to the objective,spirit and scope of the present invention. All such modifications areintended to be within the scope of the claims appended hereto. Havingnow described certain embodiments in detail, the same will be moreclearly understood by reference to the following examples, which areincluded for purposes of illustration only and are not intended to belimiting.

EXAMPLES

The following experimental examples are not intended to be limiting, andrelates to compositions and methods for generating a T cell geneknockout library by using a One-shot type II CRISPR system. One aspectincludes a method for generating a One-shot sgRNA library encoding a Tcell receptor (TCR) and/or chimeric antigen receptor (CAR) gene togetherwith sgRNA library. Another aspect includes generating modified T cellsby transduction of one-shot sgRNA library and subsequent electroporationof Cas9 endonuclease capable of altering endogenous gene expression.Also described are methods for in vitro and in vivo whole genome widegene screening, by screening two CRISPR/Cas9 T cell libraries witheither a NY-ESO-1 TCR or PSCA CAR in multiple NSG tumor mouse models.Deep sequencing of DNA from tumor infiltrating lymphocytes (TIL)isolated from treated mice identify the a panel of enriched gRNAs thattarget genes of hsa-mir-4508, C1orf141, PAGR1, CEACAM19, MFSD5, SIX2,KLF4, USP27X, CCDC33 and ZNF124. CAR-T or TCR-T cells with endogenousC1orf141, PAGR1, CEACAM19, SIX2, KLF4, USP27X or CCDC33 downregulatedwere tested in NSG mouse tumor models of prostate cancer (PC3-PASC),pancreatic cancer (CaPan1) or lung cancer (A549-ESO). Without beingbound by any theory, tt was found that PAGR1, SIX2, and Klf4downregulation could improve T cell function both in vitro and in vivoin different mouse tumor models.

Materials and Methods

Primary Human Lymphocytes.

Primary lymphocytes were stimulated with microbeads coated with CD3 andCD28 stimulatory antibodies (Life Technologies, Grand Island, N.Y.,Catalog) as described (Human gene therapy 2011, 22(12):1575-1586). Tcells were cryopreserved at day 10 in a solution of 90% fetal calf serumand 10% dimethylsulfoxide (DMSO) at 1×10⁸ cells/vial.

Propagation of Primary T Cells.

Primary human T cells were cultured in RPMI 1640 supplemented with 10%FCS, 100-U/ml penicillin, 100-g/ml streptomycin sulfate, 10-mM Hepes,and stimulated with magnetic beads coated with anti-CD3/anti-CD28 at a1:3 cell to bead ratio. Cells were counted and fed every 2 days and onceT cells appeared to rest down, as determined by both decreased growthkinetics and cell size, the T cells were either used for functionalassays or cryopreserved.

Generation of PSCA CAR One-Shot Constructs for Lentiviral Transduction.

PSCA CAR was synthesized and subcloned into Human GeCKO Lentiviral sgRNALibrary v2 (lentiGuide-Puro). The library was amplified and lentiviralvector was generated for transducing T cells.

CAR T Cell Gene Editing with One-Shot CRISPR.

Cas9 mRNA was transcribed in vitro using mMESSAGE mMACHINE T7 ULTRA kits(Life Technologies, AM1345, Carlsbad, Calif.). gRNA were transcribedusing a HiScribe™ T7 High Yield RNA Synthesis Kit. Cas9 protein waspurchased from PNA Bio (CP01). Electroporation of CRISPR reagents withone-shot CAR or CAR T cells was performed with a BTX830 electroporator.

Briefly, T cells were washed three times with OPTI-MEM and re-suspendedin OPTI-MEM (Invitrogen) at a final concentration of 1-3×10⁸ cells/ml.Subsequently, 0.1 ml of the cells was mixed with IVT RNA andelectroporated in a 2 mm cuvette. Twenty micrograms of Cas9 mRNA waselectroporated into the cells using a BTX830 (Harvard Apparatus BTX) at360 V and 1 ms. Following electroporation, the cells were immediatelyplaced in 2 ml of pre-warmed culture media and cultured in the presenceof IL-2 (100 IU/ml) at 37° C. and 5% CO₂.

Flow Cytometry.

The following monoclonal antibodies and reagents were used withindicated specificity and the appropriate isotype controls. From BDBiosciences (San Jose, Calif.): APC-conjugated anti-CD3 (555335). Datawas acquired on a FACS Accuri (BD Biosciences, San Jose, Calif.) usingCellQuest version 3.3 (BD Biosciences, San Jose, Calif.) and analyzed byFCS Express version 3.00 (De Novo Software, Los Angeles, Calif.) orFlowJo version 7.6.1 (Tree Star, Inc. Ashland, Oreg.).

Mouse Xenograft Studies.

All animal experiment protocols were approved and conducted inaccordance with the Institutional Animal Care and Use Committee. Studieswere performed as previously described with certain modifications.Briefly, for the Nalm6 tumor model, 6- to 10-week-old NSG mice wereinjected with 1×106 Nalm6 tumors cells through the tail vein on day 0.The T cell treatment began on day 7 after the tumor inoculation. T cellswere administered at a dose of 2×10⁶ cells/mouse (2M).

Example 1: Genome-Wide Functional Screening in TCR- or CAR-T Cells withCRISPR Library

Gene modified TCR- or CAR-T cell libraries comprising the one-shotCRISPR system can be used for genome wide functional screen in vitro orin vivo. Gene modified TCR- or CAR-T cell CRISPR libraries are madeusing a construct as depicted in FIG. 1A, according to an embodiment ofthe present invention. The preparation of the libraries was performedaccording to the process depicted in FIG. 1B. In vitro genome widescreening was performed as depicted in FIG. 2, and in vivo genome widescreening was performed as depicted in FIGS. 3A and 3B. FIGS. 4A and 4Bshow the fold expansion of CAR-T cell libraries stimulated in vitro withirradiated target tumor cells for a CD19-directed CAR-T cell library(FIG. 4A) and a PSCA directed CAR-T cell library (FIG. 4B). FIG. 5 showsthe functional analysis of a CD19-directed CAR-T cell library and aPSCA-directed CAR-T cell library. CD19-directed CAR-T cell library tumorinfiltrating lymphocytes (TILs) were able to eliminate Nalm6-GFP tumorcells (FIG. 5A). PSCA-directed CAR-T cell library TILs were able toupregulate the T cell activation marker CD137 after co-culture withPC3-PSCA tumor cells (FIG. 5B).

Example 2: Genome-Wide Functional Screening in CAR-T Cells with CRISPRLibrary

CAR-T cell libraries underwent in vivo selections. Briefly, selectionwas directly carried out in vivo by injecting CAR-T cells into micebearing PC3-PSCA tumor. 14 days post CAR-T cell infusion,tumor-infiltrating CAR-T cells were collected (FIG. 6).

Example 3: Deep Sequencing and Data Analysis

Preparation of barcoded DNA libraries was achieved by incorporatingbarcode and adaptor sequences into PCR primers. Miseq was performed toidentify the enrichment of each guide RNA. Over 240 guide RNAs werefound to show relative enrichment more than 5 times, including 40 guideRNAs enriched more than 10 times (FIG. 7). Top candidates identified bythe in vivo screen included Klf4, Bach, and PTGIR which were found toshow sgRNA enrichment of 50 to 100 times (FIGS. 8A and 8B). Withoutbeing bound by any theory, these results indicate that a One-shot CRISPRsystem is a potent and reliable system for in vivo screening of CAR-Tcell negative regulatory genes.

Example 4: Identifying Common CAR-T Cell Regulatory Genes AmongDifferent Tumor Types

To further verify the candidates, another in vivo screen was conductedin a native PSCA antigen bearing pancreatic cancer-CaPan1, which hasrelatively low antigen expression compared to PC3-PSCA tumor. A similarscreening approach was performed, and enriched sgRNAs were measured bydeep sequencing. The top 20 enriched candidates did not have any overlapwith the previous screen (FIG. 9).

Without being bound by any theory, these differences may be due todifferent metabolic pathway preferences, inhibitory molecules profiles,and/or micro-environments among different tumor types. By comparing thetop 100 candidates, two candidates were found to be highly enriched inboth screens: Klf4 and PAGR1. Ten overlapping candidates were foundamong the top 200 in both screens. All ten candidates were found to havebeen enriched 50 to 100 times in the PC3-PSCA model and even higher inthe CaPan1 model (FIGS. 10A and 10B). FIG. 11 shows the rank and foldchange of the top candidates.

Table 2 sets forth top 10 enriched target genes in two separateexperiments.

TABLE 2 Folds Folds Rank Rank p Rank p Rank in in in in in in Exp-1Exp-2 Exp-1 Exp-2 Exp-1 Exp-2 Hsa-mir-4508 167 134 7 5 1884 10 C1orf141121 137 10 4 106 12 PAGR1 85 66 32 13 273 35 CEACAM19 82 52 39 25 300 70MFSD5 80 42 40 38 11276 109 SIX2 74 25 53 114 47 313 KLF4 71 102 57 6111 17 USP27X 69 59 64 16 216 43 CCDC33 63 68 82 11 431 27 ZNF124 87 3529 54 2858 122

Table 3 sets forth selected candidate target genes in tumor animalmodels.

TABLE 3 Rank in Rank in Exp-3 (in Exp-4 (In Candidates vivo) vitro)PAGR1 81 163 C1orf141 11477 2138 CEACAM19 11285 6429 CCDC33 14710 812USP27X 8541 23895 SIX2 129 837 KLF4 26 302

Example 5: Candidate Validation in Tumor Models

To validate the function in tumor models, six potential candidates wereindividually knocked-out in CAR-T cells, and tested in PSCA-expressingPC3-PSCA and CaPan1 tumor mouse models. Accelerated tumor clearance wasobserved in the PAGR1, Klf4 and SIX2 KO groups (FIGS. 12A-12C). Whentested in PSCA low-expressing tumor model, PAGR1 and SIX2 KO augmentedCAR-T cell function (FIGS. 13A-13C). To test whether these candidatesenhance the function of TCR-T cells, another validation with NY-ESO-1TCR T cells in a A549-NY-ESO lung cancer was performed. PAGR1 KONY-ESO-1 TCR-T cells demonstrated better tumor control than wild typecounterparts (FIGS. 14A-14C).

Based on superior tumor clearance in the tumor models, PAGR1, Klf4, andSIX2 were further investigated. To test how these genes regulate CAR-Tcell function, functions of gene knockout CAR-T cells were tested invitro. Significant elevated degranulation was observed in all of thethree gene knockout CAR-T cells, no matter the sgRNA used (FIGS. 15A and15B). This observation is consistent with elevated tumor cytotoxicity ofthe knockout CAR-T cells, compared with the wild type control (FIGS. 15Cand 15D). The three candidates, when knocked-out, also enhanced CAR-Tcell proliferation, which was confirmed by a CSFE assay (FIG. 15E). Asecond validation was performed in PC3-PSCA and CaPan1 models. PAGR1 KOshowed superior tumor control in both models and Klf4 KO exhibitsenhanced tumor control in the PC3-PSCA model (FIGS. 16A-16C and FIGS.17A-17B).

Example 6: PAGR1 KO Enhances CAR-T Cell Tumor Accumulation andSuppression Resistance

Both PAGR1 and Klf4 KO significantly enhanced the number of PC3-PSCAtumor infiltrating CART cells. It was further found that PAGR1 KO CAR-Tcells infiltrated significantly more than wild type CAR-T cells (FIGS.18A and 18B). PAGR1 KO tumor infiltrating CAR-T cells were found todemonstrate superior tumor clearance in vitro (FIG. 18C). Without beingbound by any theory, these findings may indicate that PAGR1 KO conferredinhibitory resistance to CAR-T cells. Indeed, it was found that tumorinfiltrating PAGR1 KO CAR-T cells expressed less inhibitory moleculesthan the other groups (FIGS. 18D-18G). FIG. 18H also shows that PAGR1 KOenhances killing ability of the tumor infiltrating cells.

Example 7: PAGR1 KO Reduces Apoptosis by Epigenetic Regulation ofHistone Methylation

PAGR1 KO significantly reduced CAR-T cell apoptosis compared to wildtype CAR-T cells, either with CD3/CD28 beads stimulation or antigenexposure by co-culturing with target tumor cells (FIGS. 19A and 19B).PAGR1 was reported to be a component of the histone methylation complexHistone-lysine N-methyltransferase 2D (KMT2D). PAGR1 KO demonstratedreduced H3K4 mono-methylation and di-methylation, consistent with thephenotype of KMT2D loss-of-function in B cell lymphoma (FIG. 20A).Reduced expression of downstream genes regulated by KMT2D was observedin PAGR1 KO CAR-T cells (FIG. 20B). H3K4 mono-methylation occupancy atthe negative regulators of cell survival ARID1A and PRMT1 promoters wasfound to be greatly reduced (FIG. 20C). RNA levels of pro-survivalfactors, such as Bcl2 and Myc were found to be up-regulated (FIG. 20D).

Example 8: PAGR1 KO Enhances Adoptive T Cell Therapy in a SyngeneicModel

To test whether the downregulation of these candidates enhances thefunction of adoptive T cell therapy in an immunocompetent model, PAGR1and Klf4 KO OT-I mouse T cells were infused into B16-OVA tumor bearingmice. As confirmed by the tumor size, PAGR1 KO significantly enhancedthe function of OT-I mouse T cells than the wild type control (FIG. 21).

TABLE 4 SEQ Protein or ID Nucleic NO: Acid Sequence  1 PRT SLLMWITQC  2PRT GSGGS repeat 1-5, n at least 1  3 PRT GGGS repeat 1-4, n at least 1 4 PRT GGGGS repeat 1-5, n at least 1  5 PRT GGSG  6 PRT GGSGG  7 PRTGSGSG  8 PRT GSGGG  9 PRT GGGSG 10 PRT GSSSG 11 PRT GGGGS 12 PRTGGGGSGGGGSGGGGS 13 DNA GGTGGCGGTGGCTCGGGCGGTGGTGGGTCGGGT GGCGGCGGATCT 14PRT DKTHT 15 PRT CPPC 16 PRT CPEPKSCDTPPPCPR 17 PRT ELKTPLGDTTHT 18 PRTKSCDKTHTCP 19 PRT KCCVDCP 20 PRT KYGPPCP 21 PRT EPKSCDKTHTCPPCP 22 PRTERKCCVECPPCP 23 PRT ELKTPLGDTTHTCPRCP 24 PRT SPNMVPHAHHAQ 25 PRTEPKSCDKTYTCPPCP 26 PRT RXKR x is any amino acid 27 PRTRXRR x is any amino acid 28 PRT XRXXR x1 is R/K, x2 is any amino acid, x3 is R/K 29 PRT RXXR x is any amino acid 30 PRTRQKR x is any amino acid 31 gRNA AATCAGTATTTCCGCTGCCG target 32 gRNATTGTACCTGGGGTGCGTCTC target 33 gRNA AGGAGCAGATCCTTCGTACC target 34 gRNACCGGTAAGGCCGAGGACGAG target 35 gRNA CCCCTCGTCCTCGGCCTTAC target 36 gRNAATTGACCGGAGACGCACCCC target 37 gRNA GCGGGAATTTGCGGCGCACG target 38 gRNAACCCCGCGAGAAGCGTGAGC target 39 gRNA GAGTGGTCTGGCGTCCCCGA target 40 gRNAAACAGCCACAACCCGCTGAA target 41 gRNA TTGCTCCTGCGTGAAGCCGA target 42 gRNACAAGGCACACTACATCGAGG target 43 gRNA GTGGTGGCGCCCTACAACGG target 44 gRNAAGCCCGCGTAATCACAAGTG target 45 gRNA GCGCGGCGGCCCGCCGTTGT target 46 gRNATCTTTCTCCACGTTCGCGTC target 47 gRNA CACCCACACTTGTGATTACG target 48 gRNAGAGAAGACACTGCGTCAAGC target 49 gRNA CGCGGCGCACGACTGCGACG target 50 gRNAGTGAGATGTCGTCGCTGTTT target 51 gRNA CTCGATGCCAGTTGTAGTAT target 52 gRNAGTCCAGTACGTCCTTAATAC target 53 gRNA TCTTAAACCGATCGTAAAGC target 54 gRNAACTGCTTGCGGAGGTTTACG target 55 gRNA CTCTGAGGCCGTTGTATCCC target 56 gRNAATACAACGGCCTCAGAGGGA target 57 gRNA GATCCCTGGCCCCTCGGAGC target 58 gRNACATGTGCTGGGCGTCACTGA target 59 gRNA GGCCGCCAGGATCCCAGCGT target 60 gRNAATCCTGGCGGCCACCATCAT target 61 gRNA TCTTGCTACATCCGCGTCTA target 62 gRNACTTTGATATTGCCTTAGACG target 63 gRNA GATTCTGTTGGTCTCTTAGA target 64 gRNAAATAAAGAAAGTGAGTCAAC target 65 gRNA AAGAGACCAACAGAATCCAA target 66 gRNAACATTTGTTTGAATAAAGAA target

TABLE 5 SEQ gRNA ID name gRNA sequence NO: PAGR1 AATCAGTATTTCCGCTGCCG 31PAGR1 TTGTACCTGGGGTGCGTCTC 32 PAGR1 AGGAGCAGATCCTTCGTACC 33 PAGR1CCGGTAAGGCCGAGGACGAG 34 PAGR1 CCCCTCGTCCTCGGCCTTAC 35 PAGR1ATTGACCGGAGACGCACCCC 36 SIX2 GCGGGAATTTGCGGCGCACG 37 SIX2ACCCCGCGAGAAGCGTGAGC 38 SIX2 GAGTGGTCTGGCGTCCCCGA 39 SIX2AACAGCCACAACCCGCTGAA 40 SIX2 TTGCTCCTGCGTGAAGCCGA 41 SIX2CAAGGCACACTACATCGAGG 42 KLF4 GTGGTGGCGCCCTACAACGG 43 KLF4AGCCCGCGTAATCACAAGTG 44 KLF4 GCGCGGCGGCCCGCCGTTGT 45 KLF4TCTTTCTCCACGTTCGCGTC 46 KLF4 CACCCACACTTGTGATTACG 47 KLF4GAGAAGACACTGCGTCAAGC 48 USP27X CGCGGCGCACGACTGCGACG 49 USP27XGTGAGATGTCGTCGCTGTTT 50 USP27X CTCGATGCCAGTTGTAGTAT 51 USP27XGTCCAGTACGTCCTTAATAC 52 USP27X TCTTAAACCGATCGTAAAGC 53 USP27XACTGCTTGCGGAGGTTTACG 54 CEACAM19 CTCTGAGGCCGTTGTATCCC 55 CEACAM19ATACAACGGCCTCAGAGGGA 56 CEACAM19 GATCCCTGGCCCCTCGGAGC 57 CEACAM19CATGTGCTGGGCGTCACTGA 58 CEACAM19 GGCCGCCAGGATCCCAGCGT 59 CEACAM19ATCCTGGCGGCCACCATCAT 60 C1orf141 TCTTGCTACATCCGCGTCTA 61 C1orf141CTTTGATATTGCCTTAGACG 62 C1orf141 GATTCTGTTGGTCTCTTAGA 63 C1orf141AATAAAGAAAGTGAGTCAAC 64 C1orf141 AAGAGACCAACAGAATCCAA 65 C1orf141ACATTTGTTTGAATAAAGAA 66

Example 9: Generating DNMT3A Knockout CAR T Cells for AdoptiveImmunotherapy

Examples 9-12 describe a method of generating exhaustion-resistant Tcells for adoptive immunotherapy by knocking out the DNMT3A gene. gRNAstargeting DNMT3A were screened, and then CRISPR/Cas9 and AAV mediatedhomologous recombination was used to knockin GFP into the DNMT3A locus,which ablated the DNMT3A gene. Donor DNA comprised of EGFP andhomologous arms flanking the gRNA target, was introduced into T cellsvia AAV infection. A CD19BBz CAR was also transduced into T cells vialentivirus infection. CD19BBz+ T cells that have GFP knockin wereselected by FACS sorting and expanded in vitro. These cells exhibitedenhanced production of cytokines (IL2, INFγ, and TNFα) and degranulationwhen cocultured with cancer cells. Knockout of DNMT3A also increased theproliferation and anti-tumor effect of CD19BBz CAR T cells upon repeatedstimulation by cancer cells.

Materials and Methods

Primary Human Lymphocytes:

Primary human CD4 and CD8 T cells were isolated from healthy volunteerdonors following leukapheresis by negative selection using RosetteSepkits (Stem Cell Technologies, Vancouver BC, Canada). Primary lymphocyteswere stimulated with anti-CD3/CD28 Dynabeads (Life Technologies, GrandIsland, N.Y.).

Design and Construction of CRISPRs:

Cas9 DNA was synthesized by PCR as previously described (Cong, L. et al.(2013) Science 339, 819-823; Slaymaker, I. M. et al. (2016) Science 351,84-88) and cloned into an RNA in vitro transcription (IVT) vector, pD-Avector (Zhao, Y. et al. (2010) Cancer Research 70, 9053-9061). gRNAswere selected using web-based CRISPR algorithms (crispr.mit.edu andchopchop.rc.fas.harvard.edu). The selected sgRNAs were cloned into theMSGV vector under the control of a T7 promoter, and then synthesized byin vitro transcription. Chemically modified gRNAs (S1, S2, S3 and S4)were made by Synthego (Menlo Park, Calif.). The in vitro transcribedCas9 mRNA and sgRNAs were generated and stored as described (Ren, J. etal. (2017) Clinical cancer research, 23(9), 2255-2266).

Lentivirus and AAV Transduction:

T cells were stimulated by CD3/CD28 dynabeads on day 0, and transducedby CD19BBz lentivirus on day 1. AAV vectors were added to cells threehours after electroporation of gRNAs on day 4.

Analysis of DNMT3A Gene Editing and Knockin:

CRISPR/Cas9 gene editing was performed as previously described (Ren, J.et al. (2017) Clinical cancer research, 23(9), 2255-2266). Genomic DNAwas extracted from cells 2 or 3 days after RNA electroporation. The PCRprimers used to amplify genomic DNA fragments and analyze DNMT3Aknockout efficiencies are listed in Table 6. TIDE (Tracking of Indels byDecomposition) and ICE (Inference of CRISPR Editing) tools were used toquantify indel frequencies. Knockin was confirmed by sequencing thejunction of DNMT3A DNA and PGK-EGFP-WPRE-BGH PolyA transgene. Knockin atthe 5′ end was PCR amplified using primers CTTCTGTCACTGTTCCGGGTTTTG (SEQID NO:67) and GCCACTCCCACTGTCCTTTCCTA (SEQ ID NO:68). Knockin at the 3′end was PCR amplified using primers CACAAGGGTAGCGGCGAAGATC (SEQ IDNO:69) and ACATGCCCAGAAGCGGTGGA (SEQ ID NO:70).

TABLE 6 PCR primers used to amplify genomic DNAfragments and analyze DNMT3A knockout efficiencies Forward ReverseSequencing Exon(s) Primer Primer Primer  7 TTTCCATTTT CACCCCAATTGGAGCTCCAT TCACGGCAAG CCAGACTGC CTGAATGAGG (SEQ ID  (SEQ ID  (SEQ ID NO: 71) NO: 72) NO: 73)  8 TTTTGCTCTG ACTTCCAGGC TTTTGCTCTG TCTTGCCTCACTCCTAGTGC TCTTGCCTCA (SEQ ID  (SEQ ID  (SEQ ID  NO: 74) NO: 75) NO: 76) 9 and ACTGTATCTG CCAACAGAGAG ACTGTATCTG 10 GTCCCCTCCA CAGGTCATTCGTCCCCTCCA (SEQ ID  (SEQ ID  (SEQ ID  NO: 77) NO: 78) NO: 79) andCCAACAGAGAG CAGGTCATTC (SEQ ID  NO: 80) 11 and CTGGGGTCAG CCATTGACAGCTGGGGTCAG 12 GACTTGAATG GAGAGCAGAA GACTTGAATG (SEQ ID (SEQ ID (SEQ IDNO: 81) NO: 82) NO: 83) and     CCATTGACAG GAGAGCAGAA (SEQ ID  NO: 84)13 AGATGATGGC CAAAAGCTTG CAAAAGCTTG GTTCGAGACT AAACCCAAGG  AAACCCAAGG(SEQ ID (SEQ ID (SEQ ID NO: 85) NO: 86) NO: 87) 14 and CTGACCCTGGAGGGTCCTAA CTGACCCTGG 15 CTAAGGTGGT  GCAGTGAGCA  CTAAGGTGGT  (SEQ ID(SEQ ID (SEQ ID NO: 88) NO: 89) NO: 90) and AGGGTCCTAA GCAGTGAGCA(SEQ ID  NO: 91) 19 GACAGCTATT TGCAAAGCAG TGCAAAGCAG CCCGATGACC AAGTCACCAG  AAGTCACCAG  (SEQ ID (SEQ ID (SEQ ID NO: 92) NO: 93) NO: 94)

Constructs:

The PAAV6-MCS plasmid (Cell Biolabs, Inc) was digested by MluI and PmlI,and the 2907 bp fragment was used as the backbone. DNA fragments ofDNMT3A 5′arm, WPRE-BGH PolyA, and DNMT3A 3′ arm were synthesized by IDT.Target sequences of DNMT3A gRNAs 8-2, 8-3 and 8-4(aaggcacccgctgggtcatgtggttcggagacgg) (SEQ ID NO:95) was added to the 5′ends of both 5′ arm and 3′ arm. PGK promoter-EGFP was PCR amplified. Thefragment DNMT3A 5′arm-PGK promoter-EGFP-WPRE-BGH PolyA-DNMT3A 3′ arm wasassembled by overlapping PCR, digested by MluI and PmlI, and thenligated with PAAV6 backbone. AAV vectors were produced by Genecopoeia(Rockville, Md.).

Rapid T Cell Expansion Protocol:

0.1 million T cells were mixed with 25 million irradiated allogeneicperipheral blood mononuclear cells in 25 ml R10 medium with 30 ng/mlmouse anti-Human CD3 monoclonal antibody (Thermo Fisher Scientific16-0037-85) on day 0. 300 IU/ml Interleukin-2 was added to the cultureon day 2. 20 ml medium was replaced with fresh medium containing 300IU/ml IL-2 on day 5. Cells were split and fed with fresh mediumcontaining 300 IU/ml IL-2 on day 8 and day 11, and were cryopreserved onday 14.

Flow Cytometry:

The following monoclonal antibodies and reagents were used with theindicated specificity and the appropriate isotype controls. From BDBiosciences (San Jose, Calif.): APC-conjugated anti-CD3 (555335),FITC-anti-CD8 (555366), PE-anti-CD8 (555635), PE-anti-CD107a (555801),PECy7-anti-TNF (557647) and V450-anti-INFγ (560371). From JacksonImmunoResearch Laboratories, Inc. (West Grove, Pa.): Biotin-SPAffiniPure Goat Anti-Mouse IgG (115-065-072). From Biolegend (San Diego,Calif.): BV785-anti-CD45 (304048), AF700-anti-CD8a (300920),AF647-anti-Granzyme B (515406), and BV605-anti-IL-2 (500332).

For intracellular cytokine staining, cells were first stained usingLIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific)in order to exclude dead cells. Next, the cells were stained for surfaceantigens, fixed and permeabilized using FIX & PERM Cell Fixation & CellPermeabilization Kit (Thermo Fisher Scientific), and then stained forcytokines. Data were acquired on a Fortessa (BD Biosciences, San Jose,Calif.) and data were analyzed with FlowJo version 7.6.1 (Tree Star,Inc., Ashland, Oreg.).

Sequential Killing Assay:

0.1 million T cells were cocultured with 0.2 million K562, K562-CD19,Raji, or Nalm6 cells on Day 1. Medium was removed and 0.2 million tumorcells in fresh medium were added on Day 4 and Day 7. Cells were stainedwith LIVE/DEAD™ Fixable Aqua (Invitrogen), CD4 and CD8, and CountbrightAbsolute Counting Beads (Invitrogen) were used to count CD4 cells, CD8cells and tumor cells.

Example 10: Screening gRNAs Targeting DNMT3A

Knockout efficiencies of gRNAs targeting DNMT3A exons 7-15 and 19 wereevaluated using TIDE and ICE tools (Tables 7-10). Intracellular stainingfor DNMT3A was performed to confirm knockout efficiencies for selectedgRNAs. (FIG. 22).

TABLE 7 Sequences and gene editing efficiencies ofgRNAs targeting DNMT3A exon 7. SEQ gRNA TIDE ICE ID Name gRNA sequence %% NO: Exon 7 X2 CTCGTCATCGCCTGCTTTGG  1.3  0  96 X5 TCAGGCGTGGTAGCCACAGT 0.3  0  97 X7 TGGCTCGTCATCGCCTGCTT  0.2  0  98 X10 CTACCACGCCTGAGCCCGTG 0.7  0  99 X14 GACAAGAATGCCACCAAAGC  0.1  0 100  4 CGATGACGAGCCAGAGTACG 4.3  0 101 10 AAGCCGCTCACCTCGTACTC  1.1  0 102 30 GCTACCACGCCTGAGCCCGT14.2 16 103 31 GAGCCCGTGGGGTCCGATGC  4.2  0 104 34 GGCTACCACGCCTGAGCCCG 0.8  0 105  7.1 GGGGCCCGGGGAGTCTCAGA 10.9 106  7.2 GCCCGTGGGGTCCGATGCTG17 107 15 TGTCTTGGTGGATGACGGGC  1.5 108 original S1 TCTGAGACTCCCCGGGCCCC74.2 88 109 S2 CTCGTCATCGCCTGCTTTGG 30.1* 87 110 S3 CAGGCGTGGTAGCCACAGTG54.6 58 111 S4 GGAAGAAAACCAGGGGCCCG 61.9 83 112

TABLE 8 Sequences and gene editing efficiencies ofgRNAs targeting DNMT3A exons 8 and 9. SEQ gRNA TIDE ICE ID NamegRNA sequence % % NO: Exon 8 X3 TCCGAACCACATGACCCAGC  3.6  3 113 X4CGGAGACGGCAAATTCTCAG  2  1 114 32 GACGGCCGGGGCTTTGGCAT  2.2  1 115 33GGCCAGGCCGCATTGTGTCT  4.6  4 116 35 TGGGTCATGTGGTTCGGAGA  2  1 117 37TGGGTCATGTGGTTCGGAGA  8.1  9 118  8.1 CGGCCGGGGCTTTGGCATTG  7.4 119  8.2TGGGTCATGTGGTTCGGAGA 12.8 120  8.3 ACCCGCTGGGTCATGTGGTT  1.9 121 16TCCCCAGCATCGGACCCCAC  4.4 122 original Exon 9  9 CATGGGCTGCTTGTTGTACG 3.4  3 123 12 GCTGCTTGTTGTACGTGGCC  1.3  0 124 15 GCACTGCAAAACGAGCTCAG82.5 83 125 25 GTTTTGCAGTGCGTTCCACC  4.1  5 126 27 GCTTGTTGTACGTGGCCTGG 1.9  3 127

TABLE 9 Sequences and gene editing efficiencies ofgRNAs targeting DNMT3A exons 10, 11, and 12. SEQ gRNA TIDE ICE ID NamegRNA sequence % % NO: Exon 10 ADD1 AGAACAAGCCCATGATTGAA  2.7  8 128 13CATCGCTGTCGTGGCACACC  2.7  4 129 14 CCGGGAACAGCTTCCCCGCG  7.6  4 130 24CCCAGGGCCCATTCAATCAT  2.2  2 131 Exon 11 ADD3 AAAGCCCCGGAAGAGCACAG 36.240 132 ADD12 AGAAGTGTACACGGACATG  9.3  3 133  3 ATTATTGATGAGCGCACAAG 3.8  1 134 ADD2 AAGAAGTGTACACGGACATG  6  1 135 26 TTCTCCGCTGTGCTCTTCCG 9.7  2 136 Exon 12 ADD9 AGCGGCTGGTGTACGAGGTG  2.9  1 137 ADD10ACGAGGTGCGGCAGAAGTGC  3.7  2 138 18 TGCAGAGCGGCTGGTGTACG  2.2  3 139 29CAGAAGTGCCGGAACATTGA 14.2 11 140

TABLE 10 Sequences and gene editing efficiencies ofgRNAs targeting DNMT3A exons 13, 14, 15 and 19. SEQ gRNA TIDE ICE IDName gRNA sequence % % NO: Exon 13 16 GGCACATTCCTCCAACGAAG  0  1 141 28GCACATTCCTCCAACGAAGA 10.7 10 142 Exon 14  1 GCGTACCAGTACGACGACGA 31.1 39143  5 GGTAGCCGTCGTCGTCGTAC  2.7  3 144  8 GCGGAAACAACAACTGCTGC  2  1145 Exon 15  2 CTTCGCTAATAACCACGACC  1.2  2 146  6 TGCGGGCACAAGGGTACCTA 0.5  1 147 11 TGCTACATGTGCGGGCACAA  0.8  1 148 17 CCGCACATGTAGCAGTTCCA 0.8  1 149 19 GCGGGCACAAGGGTACCTAC 68.3 72 150 21 CACTCACAAATTCCTGGTCG 0.8  1 151 22 GGTTATTAGCGAAGAACATC  3  4 152 23 GTACCTACGGGCTGCTGCGG 9.6 17 153 Exon 19 ADD11 GCATGATGCGCGGCCCA  1.7  1 154

TABLE 11 Sequences and gene editing efficiencies ofgRNAs described in Patent WO 2017/079642 A1. SEQ gRNA TIDE ICE ID NamegRNA sequence Exon % % NO: ADD1 AGAACAAGCCCATGATTGAA Exon 10 2.7  8 155ADD2 AAGAAGTGTACACGGACATG Exon 11 6  1 156 ADD3 AAAGCCCCGGAAGAGCACAGExon 11 36.2 40 157 ADD9 AGCGGCTGGTGTACGAGGTG Exon 12 2.9  1 158 ADD10ACGAGGTGCGGCAGAAGTGC Exon 12 3.7  2 159 ADD11 GCATGATGCGCGGCCCA Exon 191.7  1 160 ADD12 AGAAGTGTACACGGACATG Exon 11 9.3  3 161

Example 11: Knockin of GFP into DNMT3A Locus in CAR T Cells

T cells were first transduced with CD19BBz lentivirus, followed byCRISPR/Cas9 gene editing at the DNMT3A locus using gRNAs 8.2 and 8.3.AAVs vector harboring DNMT3A homologous arms and EGFP (FIGS. 23-24) wereused to infect T cells and served as the template for homology-directedrepair in order to knockin EGFP. FACS was performed on day 10 to examinethe expression of CD19BBz and EGFP (FIG. 25). Untransduced cells (NTD)did not express CD19BBz and EGFP. CD19BBz cells were only transduced byCD19BBz lentivirus. 69.2% of the cells expressed CD19BBz, but they didnot express EGFP. These CAR T cells were sorted by FACS. For T cellstransduced by both lentivirus and AAV (CD19BBz+KI), 77.77% of cellsexpressed CD19BBz and 3.85% cells expressed EGFP. The 2.87% doublepositive cells were sorted by FACS. NTD, sorted CD19BBz T cells, andsorted CD19BBz+EGFP+ cells were expanded using the Rapid T cellExpansion Protocol (REP). After expansion with the REP protocol, 95% ofthe cells expressed CD19BBz in the CD19BBz group, and 87.5% of the cellsexpressed both CD19BBz and EGFP in the CD19BBz+KI group (FIG. 26).Knockin of EGFP into the DNMT3A gRNA target region was confirmed by PCRamplification of the junction between DNMT3A DNA and the transgene (FIG.27).

Example 12: Knockout of DNMT3A Enhanced the Function of CD19BBz T Cells

NTD, CD19BBz, and CD19BBz+KI T cells were cocultured with K562 cancercells that do not express CD19, and three cancer cell lines that expressCD19: K19 (Forced expression of CD19 in K562 cells), Nalm6 and Raji.CD107a was expressed at significant levels suggesting degranulation isincreased in CD19BBz+KI cells (FIG. 28). INFγ and TNFα were induced athigher levels in both CD4 and CD8 CD19BBz+KI cells (FIGS. 29-30).Although IL-2 was expressed at lower levels in CD8 cells, it wasexpressed at higher levels in CD4 CD19BBz+KI cells (FIG. 31). T cellswere repeatedly challenged by tumor cells on Day 1, Day 4 and Day 7 andthen the number of CD8 T cells and tumor cells were counted on Day 10.Significantly more CD19BBz KI T cells were present than CD19BBz T cellswhen cocultured with CD19+ tumor cells (FIG. 32), indicating knockout ofDNMT3A increased cell proliferation upon antigen stimulation. Moreimportantly, CD19BBz KI T cells almost completely eliminated Nalm6cells, while CD19BBz T cells failed to efficiently control Nalm6 cellsafter repeated stimulations (FIG. 33).

CRISPR/Cas9 gene editing technology combined with donor DNA delivery byAAV vectors successfully generated knockin of EGFP into the DNMT3A genein T cells. The EGFP+ knockin cells could be sorted and expanded invitro, and they exhibited increased cytokine production, proliferationand cytotoxicity in response to cancer cells. DNMT3A is required for denovo DNA methylation that is involved in the development of T cellexhaustion. This approach created DNMT3A knockout CAR T cells that havestronger anti-tumor function.

Other Embodiments

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1. A modified immune cell or precursor cell thereof, comprising: aninsertion and/or deletion in a gene locus encoding for a transcriptionalmodulator, wherein the insertion and/or deletion is capable ofdownregulating gene expression of the endogenous transcriptionalmodulator; and an exogenous T cell receptor (TCR) and/or chimericantigen receptor (CAR) comprising affinity for an antigen on a targetcell.
 2. The modified cell of claim 1, optionally wherein: the insertionand/or deletion in a gene locus is mediated by a CRISPR-related systemand/or the insertion and/or deletion in a gene locus is mediated byCRISPR/Cas9.
 3. (canceled)
 4. The modified cell of claim 1, optionallywherein the transcriptional modulator is a transcription factor or anepigenetic regulator, optionally wherein: the transcription factor isSIX2 or KLF4, optionally wherein: the insertion and/or deletion in thegene locus encoding for SIX2 is capable of downregulating geneexpression of SIX2, and/or downregulating gene expression of one or moredownstream targets of SIX2, or the insertion and/or deletion in the genelocus encoding for KLF4 is capable of downregulating gene expression ofKLF4, and/or downregulating gene expression of one or more downstreamtargets of KLF4; or the epigenetic regulator is a modulator of histonemethylation, optionally wherein: the modulator of histone methylation isa component of a histone methyltransferase complex, optionally whereinthe component of a histone methyltransferase complex ishistone-lysine-N-methyltransferase 2D (KMT2D) or PAGR1. 5.-13.(canceled)
 14. The modified cell of claim 4, wherein the insertionand/or deletion in the gene locus encoding for PAGR1 is capable ofdownregulating gene expression of PAGR1, and/or downregulating geneexpression of one or more downstream targets of the PAGR1-associatedhistone methyltransferase complex, optionally wherein the one or moredownstream targets of the PAGR1-associated histone methyltransferasecomplex is selected from the group consisting of ARID1A, ARID3B, ASXL1,DNMT3A, DUSP1, MAP3K8, PAXIP1, PRMT1, SOCS3, and TNFAIP3.
 15. (canceled)16. The modified cell of claim 1, optionally wherein: the exogenous TCRis selected from the group consisting of a wild-type TCR, a highaffinity TCR, and a chimeric TCR; the exogenous TCR comprises at leastone disulfide bond; the exogenous TCR comprises a TCR alpha chain and aTCR beta chain; and/or the exogenous CAR comprises an antigen-bindingdomain, a transmembrane domain, and an intracellular domain, optionallyfurther comprising a hinge domain, optionally wherein the hinge domainis selected from the group consisting of an Fc fragment of an antibody,a hinge region of an antibody, a CH2 region of an antibody, a CH3 regionof an antibody, an artificial hinge domain, a hinge comprising an aminoacid sequence of CD8, or any combination thereof, optionally wherein:the antigen-binding domain is selected from the group consisting of anantibody, an scFv, and a Fab; the transmembrane domain is selected fromthe group consisting of an artificial hydrophobic sequence andtransmembrane domain of a type I transmembrane protein, an alpha, beta,or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5,CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, andCD154; the intracellular domain comprises at least one co-stimulatorydomain selected from the group consisting of co-stimulatory domains ofproteins in the TNFR superfamily, CD28, 4-1BB (CD137), OX40 (CD134),PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1,Lck, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3; and/orthe intracellular domain comprises an intracellular domain selected fromthe group consisting of cytoplasmic signaling domains of a human CD3zeta chain, FcyRIII, FcsRI, a cytoplasmic tail of an Fc receptor, animmunoreceptor tyrosine-based activation motif (ITAM) bearingcytoplasmic receptors, TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. 17.-25.(canceled)
 26. A modified immune cell or precursor cell thereof,comprising an exogenous T cell receptor (TCR) and/or chimeric antigenreceptor (CAR) comprising affinity for an antigen on a target cell, andan insertion and/or deletion in one or more gene loci encoding for aprotein selected from the group consisting of AZI2, C1orf141, CCDC33,CCL7, CEACAM19, KLF4, MFSD5, PAGR1, SIX2, and USP27X, wherein theinsertion and/or deletion is capable of downregulating gene expressionof the one or more endogenous genes; an insertion and/or deletion in oneor more gene loci encoding for a protein selected from the groupconsisting of C1orf141, CCDC33, CCL7, CEACAM19, KLF4, MFSD5, PAGR1,SIX2, and USP27X, wherein the insertion and/or deletion is capable ofdownregulating gene expression of the one or more endogenous genes; aninsertion and/or deletion in one or more gene loci encoding for aprotein selected from the group consisting of KLF4, PAGR1, and SIX2,wherein the insertion and/or deletion is capable of downregulating geneexpression of the one or more endogenous genes; an insertion and/ordeletion in a gene locus encoding for KLF4, wherein the insertion and/ordeletion is capable of downregulating gene expression of endogenousKLF4; an insertion and/or deletion in a gene locus encoding for SIX2,wherein the insertion and/or deletion is capable of downregulating geneexpression of endogenous SIX2; or an insertion and/or deletion in a genelocus encoding for PAGR1, wherein the insertion and/or deletion iscapable of downregulating gene expression of endogenous PAGR1. 27.-31.(canceled)
 32. The modified cell of claim 1, optionally wherein: theantigen on a target cell is a tumor associated antigen (TAA); themodified cell is an autologous cell; the modified cell is derived from ahuman; and/or the modified cell is a modified T cell. 33.-35. (canceled)36. A method for generating a modified immune cell or precursor cellthereof, comprising: a) introducing into the immune cell a first nucleicacid comprising a nucleic acid sequence encoding an exogenous T cellreceptor (TCR) and/or a chimeric antigen receptor (CAR) comprisingaffinity for an antigen on a target cell; and b) introducing into theimmune cell one or more polypeptides and/or nucleic acids capable ofdownregulating gene expression of an endogenous transcriptionalmodulator.
 37. The method of claim 36, optionally wherein: theendogenous transcriptional modulator is a transcription factor or anepigenetic regulator optionally wherein the transcription factor is SIX2or KLF4, optionally wherein: downregulating gene expression of thetranscription factor results in downregulated gene expression of SIX2,and/or downregulated gene expression of one or more downstream targetsof SIX2, or downregulating gene expression of the transcription factorresults in downregulated gene expression of KLF4, and/or downregulatedgene expression of one or more downstream targets of KLF4; theepigenetic regulator is a modulator of histone methylation, optionallywherein the modulator of histone methylation is a component of a histonemethyltransferase complex, optionally wherein the component of a histonemethyltransferase complex is a histone-lysine-N-methyltransferase 2D(KMT2D) or PAGR1, optionally wherein downregulating gene expression ofthe component of a histone methyltransferase complex results indownregulated gene expression of PAGR1 and/or downregulated geneexpression of one or more downstream targets of the PAGR1-associatedhistone methyltransferase complex, optionally wherein the one or moredownstream targets of the PAGR1-associated histone methyltransferasecomplex is selected from the group consisting of ARID1A, ARID3B, ASXL1,DNMT3A, DUSP1, MAP3K8, PAXIP1, PRMT1, SOCS3, and TNFAIP3. 38.-46.(canceled)
 47. A method for generating a modified immune cell orprecursor cell thereof, comprising introducing into the immune cell afirst nucleic acid comprising a nucleic acid sequence encoding anexogenous T cell receptor (TCR) and/or a chimeric antigen receptor (CAR)comprising affinity for an antigen on a target cell; and conducting astep selected from the group consisting of (a) introducing into theimmune cell one or more polypeptides and/or nucleic acids capable ofdownregulating gene expression of one or more endogenous genes selectedfrom the group consisting of AZI2, C1orf141, CCDC33, CCL7, CEACAM19,KLF4, MFSD5, PAGR1, SIX2, and USP27X; (b) introducing into the immunecell one or more polypeptides and/or nucleic acids capable ofdownregulating gene expression of one or more endogenous genes selectedfrom the group consisting of C1orf141, CCDC33, CCL7, CEACAM19, KLF4,MFSD5, PAGR1, SIX2, and USP27X; (c) introducing into the immune cell oneor more polypeptides and/or nucleic acids capable of downregulating geneexpression of one or more endogenous genes selected from the groupconsisting of KLF4, PAGR1, and SIX2; (d) introducing into the immunecell one or more polypeptides and/or nucleic acids capable ofdownregulating gene expression of endogenous KLF4; (e) introducing intothe immune cell one or more polypeptides and/or nucleic acids capable ofdownregulating gene expression of endogenous SIX2; and (f) introducinginto the immune cell one or more polypeptides and/or nucleic acidscapable of downregulating gene expression of endogenous PAGR1. 48.-52.(canceled)
 53. The method of claim 36, optionally wherein: the firstnucleic acid is introduced by viral transduction, optionally wherein theviral transduction comprises contacting the cell with a viral vectorcomprising the first nucleic acid, optionally wherein the viral vectoris selected from the group consisting of a retroviral vector, alentiviral vector, an adenoviral vector, and an adeno-associated viralvector; each of the one or more polypeptides and/or nucleic acidscapable of downregulating gene expression comprises a CRISPR-relatedsystem, optionally wherein the CRISPR-related system comprises a CRISPRnuclease and a guide RNA, optionally wherein the guide RNA comprises aguide sequence that is sufficiently complementary to a target sequenceof an endogenous gene, optionally wherein the CRISPR nuclease and theguide RNA comprise a ribonucleoprotein (RNP) complex or the CRISPRnuclease and/or the guide RNA are encoded by a polynucleotide,optionally wherein the polynucleotide comprises a vector and/or asynthetic mRNA. 54.-59. (canceled)
 60. The method of claim 53,optionally wherein: the target sequence is within the PAGR1 gene andcomprises a nucleic acid sequence set forth in any one of SEQ ID NOs:31-36; the target sequence is within the SIX2 gene and wherein the guideRNA comprises a nucleic acid sequence set forth in any one of SEQ IDNOs:37-42; the target sequence is within the Klf4 gene and wherein theguide RNA comprises a nucleic acid sequence set forth in any one of SEQID NOs:43-48; the target sequence is within the USP27X gene and whereinthe guide RNA comprises a nucleic acid sequence set forth in any one ofSEQ ID NOs:49-54; the target sequence is within the CEACAM19 gene andwherein the guide RNA comprises a nucleic acid sequence set forth in anyone of SEQ ID NOs:55-60; the target sequence is within the C1orf141 geneand wherein the guide RNA comprises a nucleic acid sequence set forth inany one of SEQ ID NOs:61-66; or the guide sequence comprises a nucleicacid sequence set forth in any one of SEQ ID NOs: 31-66. 61.-68.(canceled)
 69. The method of claim 36, optionally wherein: each of theone or more polypeptides and/or nucleic acids capable of downregulatinggene expression is introduced by electroporation; the antigen on atarget cell is a tumor associated antigen (TAA); the modified cell is anautologous cell; the modified cell is derived from a human; and/or themodified cell is a modified T cell. 70.-73. (canceled)
 74. A method forenhancing a function of a modified immune cell or precursor cellthereof, wherein the modified cell comprises an exogenous T cellreceptor (TCR) and/or chimeric antigen receptor (CAR) comprisingaffinity for an antigen on a target cell, comprising: introducing intothe immune cell one or more polypeptides and/or nucleic acids capable ofdownregulating gene expression of an endogenous transcriptionalmodulator; introducing into the immune cell one or more polypeptidesand/or nucleic acids capable of downregulating gene expression of one ormore endogenous genes selected from the group consisting of AZI2,C1orf141, CCDC33, CCL7, CEACAM19, KLF4, MFSD5, PAGR1, SIX2, and USP27X;introducing into the immune cell one or more polypeptides and/or nucleicacids capable of downregulating gene expression of one or moreendogenous genes selected from the group consisting of C1orf141, CCDC33,CCL7, CEACAM19, KLF4, MFSD5, PAGR1, SIX2, and USP27X; introducing intothe immune cell one or more polypeptides and/or nucleic acids capable ofdownregulating gene expression of one or more endogenous genes selectedfrom the group consisting of KLF4, PAGR1, and SIX2; introducing into theimmune cell one or more polypeptides and/or nucleic acids capable ofdownregulating gene expression of endogenous KLF4; introducing into theimmune cell one or more polypeptides and/or nucleic acids capable ofdownregulating gene expression of endogenous SIX2; or introducing intothe immune cell one or more polypeptides and/or nucleic acids capable ofdownregulating gene expression of endogenous PAGR1. 75.-80. (canceled)81. The method of claim 74, optionally wherein: the function is tumorinfiltration; the function is tumor killing; the function is resistanceto immunosuppression; the function is resistance to immunosuppression byPD-1, LAG-3, TIM-3, and/or CTLA-4; the antigen on a target cell is atumor associated antigen (TAA); the modified cell is an autologous cell;the modified cell is derived from a human; and/or the modified cell is amodified T cell. 82.-84. (canceled)
 85. A method for inhibitingactivation-induced cell death of a modified immune cell or precursorcell thereof, wherein the modified cell comprises an exogenous T cellreceptor (TCR) and/or a chimeric antigen receptor (CAR) comprisingaffinity for an antigen on a target cell, comprising: introducing intothe immune cell one or more polypeptides and/or nucleic acids capable ofdownregulating gene expression of an endogenous transcriptionalmodulator; introducing into the immune cell one or more polypeptidesand/or nucleic acids capable of downregulating gene expression of one ormore endogenous genes selected from the group consisting of AZI2,C1orf141, CCDC33, CCL7, CEACAM19, KLF4, MFSD5, PAGR1, SIX2, and USP27X;introducing into the immune cell one or more polypeptides and/or nucleicacids capable of downregulating gene expression of one or moreendogenous genes selected from the group consisting of C1orf141, CCDC33,CCL7, CEACAM19, KLF4, MFSD5, PAGR1, SIX2, and USP27X; introducing intothe immune cell one or more polypeptides and/or nucleic acids capable ofdownregulating gene expression of one or more endogenous genes selectedfrom the group consisting of KLF4, PAGR1, and SIX2; introducing into theimmune cell one or more polypeptides and/or nucleic acids capable ofdownregulating gene expression of endogenous KLF4; introducing into theimmune cell one or more polypeptides and/or nucleic acids capable ofdownregulating gene expression of endogenous SIX2; or introducing intothe immune cell one or more polypeptides and/or nucleic acids capable ofdownregulating gene expression of endogenous PAGR1. 86.-95. (canceled)96. A method for identifying a gene that when downregulated, results inan enhanced function of an immune cell or precursor cell thereof,comprising the steps of: a) introducing into a plurality of immune cellsa library of nucleic acids encoding for an exogenous T cell receptor(TCR) and/or a chimeric antigen receptor (CAR) having affinity for anantigen, thereby generating a plurality of modified immune cells; b)introducing into the plurality of modified immune cells a plurality ofagents that target a plurality of endogenous genes, thereby generating aplurality of edited immune cells; c) contacting the plurality of editedimmune cells with a tumor cell; d) selecting one or more edited immunecells that exhibit an enhanced function of an immune cell; and e)identifying the endogenous gene that is downregulated in the one or moreedited immune cells of step d), thereby identifying the gene that whendownregulated, results in an enhanced function of an immune cell orprecursor cell thereof.
 97. The method of claim 96, optionally wherein:the steps of a) and b) are carried out simultaneously; the plurality ofagents in step b) each comprise a nucleic acid encoding a unique guideRNA that targets each of the plurality of endogenous genes, optionallywherein the identifying step e) comprises identifying the unique guideRNA that targets the downregulated gene; the plurality of agents in stepb) each comprise a CRISPR nuclease polypeptide or a nucleic acid thatencodes for a CRISPR nuclease; and/or the plurality of agents in step b)each comprise a CRISPR-related system, optionally wherein theCRISPR-related system comprises a CRISPR nuclease and a guide RNA,optionally wherein the guide RNA comprises a guide sequence that issufficiently complementary with a target sequence of a gene thatregulates a function of the immune cell, optionally wherein the CRISPRnuclease and the guide RNA comprise a ribonucleoprotein (RNP) complex.98.-104. (canceled)
 105. A method for identifying a gene that whendownregulated, results in an enhanced function of an immune cell orprecursor cell thereof, comprising the steps of: a) introducing into aplurality of immune cells a library of nucleic acids encoding for anexogenous T cell receptor (TCR) and/or a chimeric antigen receptor (CAR)having affinity for an antigen, and encoding for a plurality of guideRNAs that each target a unique region of each of a plurality ofendogenous genes, thereby generating a plurality of modified immunecells; b) introducing into the plurality of modified immune cells aCRISPR nuclease or a nucleic acid that encodes for a CRISPR nuclease,thereby generating a plurality of edited immune cells wherein geneexpression of the plurality of endogenous genes is downregulated; c)contacting the plurality of edited immune cells with a tumor cell; d)selecting one or more edited immune cells that exhibit an enhancedfunction of an immune cell; and e) identifying the endogenous gene thatis downregulated in the one or more edited immune cells of step d),thereby identifying the gene that when downregulated, results in anenhanced function of an immune cell or precursor cell thereof.
 106. Themethod of claim 96, wherein the step of contacting c) comprises:contacting the plurality of edited immune cells with a tumor cell linecomprising the tumor cell, or administering the plurality of editedimmune cells into a tumor-bearing organism comprising the tumor cell.107. (canceled)
 108. A nucleic acid library comprising one or morenucleic acids, wherein each of the one or more nucleic acids comprise: afirst nucleic acid encoding for a unique guide RNA; and a second nucleicacid encoding for an exogenous T cell receptor (TCR) and/or chimericantigen receptor (CAR) having affinity for an antigen.
 109. The nucleicacid library of claim 108, optionally wherein: the first nucleic acidcomprises guide sequences that are sufficiently complementary withtarget sequences of an endogenous gene; and/or each of the one or morenucleic acids is a vector, optionally wherein each of the vectorscomprise a first expression cassette comprising a first promoteroperably linked to the first nucleic acid, and a second expressioncassette comprising a second promoter operably linked to the secondnucleic acid, optionally wherein the second expression cassette furthercomprises a polynucleotide sequence that encodes for a selectablemarker, optionally wherein the selectable marker is a fluorescentprotein. 110.-113. (canceled)